Cerebellar granule (CG) neurons express a G protein-gated K+ current (GIRK) that is involved in the neurotransmitter regulation of the excitatory input to the Purkinje fibres of the cerebellum. Here, we characterized the single-channel behaviour of GIRK in CG neurons, and examined the effects of several known modulators of GIRK and their putative physiological roles. Whole-cell GIRKs were activated by baclofen, a GABAB receptor agonist. In cell-attached patches, baclofen activated GIRK with a single-channel conductance of 34 pS and a mean open time of 0.5 ms. In inside-out patches, application of GTPgammaS to the cytoplasmic side activated GIRK with similar kinetic properties. Addition of 2 mM ATP resulted in a marked increase in GIRK activity and induced longer-lived openings with a mean open time of 2.3 ms (ATP-dependent gating). Brain cytosolic fraction or free fatty acids inhibited this effect of ATP, and this was reversed by addition of purified recombinant brain fatty acid binding protein. Applying phosphatidylinositol 4,5-bisphosphate (PIP2) to inside-out patches in place of ATP also increased GIRK activity; however, only an increase in the frequency of opening was observed. The stimulatory effect of PIP2 on GIRK activity was not inhibited by the cytosolic fraction. Following maximal activation by PIP2, ATP caused an additional 2.2-fold increase in GIRK activity. These results show that GIRKs in CG neurons are regulated by positive and negative modulators that affect frequency as well as open time duration. The net effect is that the ligand-activated GIRK is in the 'low activity' state associated with short-lived openings, mainly due to strong action of the cytosolic inhibitor of ATP-dependent gating. Our results also show that intracellular ATP modulates GIRK via pathways different from that of PIP2 in CG neurons.

Numerous studies have demonstrated that aged black garlic (ABG) has strong anti-oxidant activity. Little is known however regarding the anti-inflammatory activity of ABG. This study was performed to identify and compare the anti-oxidant and anti-inflammatory effects of ABG extract (ABGE) with those of fresh raw garlic (FRG) extract (FRGE). In addition, we investigated which components are responsible for the observed effects. Hydrogen peroxide (H2O2) and lipopolysaccharide (LPS) were used as a pro-oxidant and pro-inflammatory stressor, respectively. ABGE showed high ABTS and DPPH radical scavenging activities and low ROS generation in RAW264.7 cells compared with FRGE. However, inhibition of cyclooxygenase-2 and 5-lipooxygenase activities by FRGE was stronger than that by ABGE. FRGE reduced PGE₂, NO, IL-6, IL-1β, LTD₄, and LTE₄ production in LPS-activated RAW264.7 cells more than did ABGE. The combination of FRGE and sugar (galactose, glucose, fructose, or sucrose), which is more abundant in ABGE than in FRGE, decreased the anti-inflammatory activity compared with FRGE. FRGE-induced inhibition of NF-κB activation and pro-inflammatory gene expression was blocked by combination with sugars. The lower anti-inflammatory activity in ABGE than FRGE could result from the presence of sugars. Our results suggest that ABGE might be helpful for the treatment of diseases mediated predominantly by ROS.

Obesity due to an excessive intake of nutrient disturbs the hypothalamus-mediated energy metabolism subsequently develops metabolic disorders. In this study, we investigated the effect of pine needle extract (PNE) on the hypothalamic proopiomelanocortin (POMC) neurons involved in the regulation of energy balance via melanocortin system and fat tissue metabolism.

Depression is more common in women with breast cancer than the general population. Selective serotonin reuptake inhibitors (SSRIs), a group of antidepressants, are widely used for the treatment of patients with depression and a range of anxiety-related disorders. The association between the use of antidepressant medication and breast cancer is controversial. In this study, we investigated whether and how SSRIs induce the death of human breast cancer MCF-7 cells. Of the antidepressants tested in this study (amitriptyline, bupropion, fluoxetine, paroxetine, and tianeptine), paroxetine most reduced the viability of MCF-7 cells in a time-and dose-dependent manner. The exposure of MCF-7 cells to paroxetine resulted in mitochondrion-mediated apoptosis, which is assessed by increase in the number of cells with sub-G1 DNA content, caspase-8/9 activation, poly (ADP-ribose) polymerase cleavage, and Bax/Bcl-2 ratio and a reduction in the mitochondrial membrane potential. Paroxetine increased a generation of reactive oxygen species (ROS), intracellular Ca2+ levels, and p38 MAPK activation. The paroxetine-induced apoptotic events were reduced by ROS scavengers and p38 MAPK inhibitor, and the paroxetine's effect was dependent on extracellular Ca2+ level. Paroxetine also showed a synergistic effect on cell death induced by chemotherapeutic drugs in MCF-7 and MDA-MB-231 cells. Our results showed that paroxetine induced apoptosis of human breast cancer MCF-7 cells through extracellular Ca2+-and p38 MAPK-dependent ROS generation. These results suggest that paroxetine may serve as an anticancer adjuvant to current cancer therapies for breast cancer patients with or without depression.

Accumulative alcohol hangovers cause liver damage through oxidative and inflammatory stress. Numerous antioxidant and anti-inflammatory reagents have been developed to reduce alcohol hangovers, but these reagents are still insignificant and have limitations in that they can cause liver toxicity. Oyster hydrolysate (OH), another reagent that has antioxidant and anti-inflammatory activity, is a product extracted through an enzymatic hydrolysis process from oysters (Crassostrea gigas), which can be easily eaten in meals. This study was aimed at determining the effects of OH on alcohol metabolism, using a single high dose of ethanol (EtOH) administered to rodents, by monitoring alcohol metabolic enzymes, oxidative stress signals, and inflammatory mediators. The effect of tyrosine-alanine (YA) peptide, a main component of OH, on EtOH metabolism was also identified. In vitro experiments showed that OH pretreatment inhibited EtOH-induced cell death, oxidative stress, and inflammation in liver cells and macrophages. In vivo experiments showed that OH and YA pre-administration increased alcohol dehydrogenase, aldehyde dehydrogenase, and catalase activity in EtOH binge treatment. In addition, OH pre-administration alleviated CYP2E1 activity, ROS production, apoptotic signals, and inflammatory mediators in liver tissues. These results showed that OH and YA enhanced EtOH metabolism and had a protective effect against acute alcohol liver damage. Our findings offer new insights into a single high dose of EtOH drinking and suggest that OH and YA could be used as potential marine functional foods to prevent acute alcohol-induced liver damage.

TWIK-related K(+) channel-2 (TREK-2) and TWIK-related spinal cord K(+) (TRESK) channel are members of two-pore domain K(+) channel family. They are well expressed and help to set the resting membrane potential in sensory neurons. Modulation of TREK-2 and TRESK channels are involved in the pathogenesis of pain, and specifi c activators of TREK-2 and TRESK may be benefi cial for the treatment of pain symptoms. However, the effect of commonly used analgesics on TREK-2 and TRESK channels are not known. Here, we investigated the effect of analgesics on TREK-2 and TRESK channels. The effects of analgesics were examined in HEK cells transfected with TREK-2 or TRESK. Amitriptyline, citalopram, escitalopram, and fluoxetine significantly inhibited TREK-2 and TRESK currents in HEK cells (p<0.05, n=10). Acetaminophen, ibuprofen, nabumetone, and bupropion inhibited TRESK, but had no effect on TREK-2. These results show that all analgesics tested in this study inhibit TRESK activity. Further study is needed to identify the mechanisms by which the analgesics modulate TREK-2 and TRESK differently.

The two-pore domain K+ (K2P) channel, which is involved in setting the resting membrane potential in neurons, is an essential target for receptor agonists. Activation of the γ-aminobutyric acid (GABA) receptors (GABAAR and GABABR) reduces cellular excitability through Cl- influx and K+ efflux in neurons. Relatively little is known about the link between GABAAR and the K+ channel. The present study was performed to identify the effect of GABAR agonists on K2P channel expression and activity in the neuroblastic B35 cells that maintain glutamic acid decarboxylase (GAD) activity and express GABA. TASK and TREK/TRAAK mRNA were expressed in B35 cells with a high level of TREK-2 and TRAAK. In addition, TREK/TRAAK proteins were detected in the GABAergic neurons obtained from GABA transgenic mice. Furthermore, TREK-2 mRNA and protein expression levels were markedly upregulated in B35 cells by GABAAR and GABABR agonists. In particular, muscimol, a GABAAR agonist, significantly increased TREK-2 expression and activity, but the effect was reduced in the presence of the GABAAR antagonist bicuculine or TREK-2 inhibitor norfluoxetine. In the whole-cell and single-channel patch configurations, muscimol increased TREK-2 activity, but the muscimol effect disappeared in the N-terminal deletion mutant. These results indicate that muscimol directly induces TREK-2 activation through the N-terminus and suggest that muscimol can reduce cellular excitability by activating the TREK-2 channel and by inducing Cl- influx in GABAergic neurons.

TWIK (tandem-pore domain weak inward rectifying K+)-related spinal cord K+ channel (TRESK), a member of the two-pore domain K+ channel family, is abundantly expressed in dorsal root ganglion (DRG) neurons. It is well documented that TRESK expression is changed in several models of peripheral nerve injury, resulting in a shift in sensory neuron excitability. However, the role of TRESK in the model of spinal cord injury (SCI) has not been fully understood. This study investigates the role of TRESK in a thoracic spinal cord contusion model, and in transgenic mice overexpressed with the TRESK gene (TGTRESK). Immunostaining analysis showed that TRESK was expressed in the dorsal and ventral neurons of the spinal cord. The TRESK expression was increased by SCI in both dorsal and ventral neurons. TRESK mRNA expression was upregulated in the spinal cord and DRG isolated from the ninth thoracic (T9) spinal cord contusion rats. The expression was significantly upregulated in the spinal cord below the injury site at acute time points (6, 24, and 48 h) after SCI (p < 0.05). In addition, TRESK expression was markedly increased in DRGs below and adjacent to the injury site. TRESK was expressed in inflammatory cells. In addition, the number and fluorescence intensity of TRESK-positive neurons increased in the dorsal and ventral horns of the spinal cord after SCI. TGTRESK SCI mice showed faster paralysis recovery and higher mechanical threshold compared to wild-type (WT)-SCI mice. TGTRESK mice showed lower TNF-α concentrations in the blood than WT mice. In addition, IL-1β concentration and apoptotic signals in the caudal spinal cord and DRG were significantly decreased in TGTRESK SCI mice compared to WT-SCI mice (p < 0.05). These results indicate that TRESK upregulated following SCI contributes to the recovery of paralysis and mechanical pain threshold by suppressing the excitability of motor and sensory neurons and inflammatory and apoptotic processes.

Tandem pore domain weak inward rectifier potassium channel (TWIK)-related spinal cord K⁺ (TRESK; K2P18.1) channel is the only member of the two-pore domain K⁺ (K2P) channel family that is activated by an increase in intracellular Ca2+ concentration ([Ca2+]i) and linked to migraines. This study was performed to identify the effect of verapamil, which is an L-type Ca2+ channel blocker and a prophylaxis for migraines, on the TRESK channel in trigeminal ganglion (TG) neurons, as well as in a heterologous system. Single-channel and whole-cell currents were recorded in TG neurons and HEK-293 cells transfected with mTRESK using patch-clamping techniques. In TG neurons, changes in [Ca2+]i were measured using the fluo-3-AM Ca2+ indicator. Verapamil, nifedipine, and NiCl₂ inhibited the whole-cell currents in HEK-293 cells overexpressing mTRESK with IC50 values of 5.2, 54.3, and >100 μM, respectively. The inhibitory effect of verapamil on TRESK channel was also observed in excised patches. In TG neurons, verapamil (10 μM) inhibited TRESK channel activity by approximately 76%. The TRESK channel activity was not dependent on the presence of extracellular Ca2+. In addition, the inhibitory effect of verapamil on the TRESK channel remained despite the absence of extracellular Ca2+. These findings show that verapamil inhibits the TRESK current independently of the blockade of Ca2+ influx in TG neurons. Verapamil will be able to exert its pharmacological effects by modulating TRESK, as well as Ca2+ influx, in TG neurons in vitro. We suggest that verapamil could be used as an inhibitor for identifying TRESK channel in TG neurons.

Sea hare-derived compounds induce macrophage activation and reduce asthmatic parameters in mouse models of allergic asthma. These findings led us to study the role of sea hare hydrolysates (SHH) in cancer pathophysiology. SHH treatment-induced M1 macrophage activation in RAW264.7 cells, peritoneal macrophages, and THP-1 cells, as did lipopolysaccharide (LPS) (+ INF-γ), whereas SHH reduced interleukin (IL)-4 (+IL-13)-induced M2 macrophage polarization. In addition, SHH treatment inhibited the actions of M1 and M2 macrophages, which have anticancer and pro-cancer effects, respectively, in non-small cell lung cancer cells (A549 and HCC-366) and tumor-associated macrophages (TAMs). Furthermore, SHH induced G2/M phase arrest and cell death in A549 cells. SHH also downregulated STAT3 activation in macrophages and A549 cells, and the down-regulation was recovered by colivelin, a STAT3 activator. SHH-induced reduction of M2 polarization and tumor growth was blocked by colivelin treatment. SHH-induced cell death did not occur in the manner of apoptotic signaling pathways, while the death pattern was mediated through pyroptosis/necroptosis, which causes membrane rupture, formation of vacuoles and bleb, activation of caspase-1, and secretion of IL-1β in SHH-treated A549 cells. However, a combination of SHH and colivelin blocked caspase-1 activation. Z-YVAD-FMK and necrostatin-1, pyrotosis and necroptosis inhibitors, attenuated SHH's effect on the cell viability of A549 cells. Taken together, SHH showed anticancer effects through a cytotoxic effect on A549 cells and a regulatory effect on macrophages in A549 cells. In addition, the SHH-induced anticancer effects were mediated by non-apoptotic regulated cell death pathways under STAT3 inhibition. These results suggest that SHH may be offered as a potential remedy for cancer immunotherapy.

The modulation of K+ channels plays a crucial role in cell migration and proliferation, but the effect of K+ channels on human cutaneous wound healing (CWH) remains underexplored. This study aimed to determine the necessity of modulating K+ channel activity and expression for human CWH. The use of 25 mM KCl as a K+ channel blocker markedly improved wound healing in vitro (in keratinocytes and fibroblasts) and in vivo (in rat and porcine models). K+ channel blockers, such as quinine and tetraethylammonium, aided in vitro wound healing, while Ba2+ was the exception and did not show similar effects. Single-channel recordings revealed that the Ba2+-insensitive large conductance Ca2+-activated K+ (BKCa) channel was predominantly present in human keratinocytes. NS1619, an opener of the BKCa channel, hindered wound healing processes like proliferation, migration, and filopodia formation. Conversely, charybdotoxin and iberiotoxin, which are BKCa channel blockers, dramatically enhanced these processes. The downregulation of BKCa also improved CWH, whereas its overexpression impeded these healing processes. These findings underscore the facilitative effect of BKCa channel suppression on CWH, proposing BKCa channels as potential molecular targets for enhancing human cutaneous wound healing.

The influence of spinal cord injury (SCI) on protein expression in the rat urinary bladder was assessed by proteomic analysis at different time intervals post-injury. After contusion SCI between T9 and T10, bladder tissues were processed by 2-DE and MALDI-TOF/MS at 6 hr to 28 days after SCI to identify proteins involved in the healing process of SCI-induced neurogenic bladder. Approximately 1,000 spots from the bladder of SCI and sham groups were visualized and identified. At one day after SCI, the expression levels of three protein were increased, and seven spots were down-regulated, including heat shock protein 27 (Hsp27) and heat shock protein 20 (Hsp20). Fifteen spots such as S100-A11 were differentially expressed seven days post-injury, and seven proteins including transgelin had altered expression patterns 28 days after injury. Of the proteins with altered expression levels, transgelin, S100-A11, Hsp27 and Hsp20 were continuously and variably expressed throughout the entire post-SCI recovery of the bladder. The identified proteins at each time point belong to eight functional categories. The altered expression patterns identified by 2-DE of transgelin and S100-A11 were verified by Western blot. Transgelin and protein S100-A11 may be candidates for protein biomarkers in the bladder healing process after SCI.

Cells can resist and even recover from stress induced by acute hypoxia, whereas chronic hypoxia often leads to irreversible damage and eventually death. Although little is known about the response(s) to acute hypoxia in neuronal cells, alterations in ion channel activity could be preferential. This study aimed to elucidate which channel type is involved in the response to acute hypoxia in rat pheochromocytomal (PC12) cells as a neuronal cell model. Using perfusing solution saturated with 95% N(2) and 5% CO(2), induction of cell hypoxia was confirmed based on increased intracellular Ca(2+) with diminished oxygen content in the perfusate. During acute hypoxia, one channel type with a conductance of about 30 pS (2.5 pA at -80 mV) was activated within the first 2~3 min following onset of hypoxia and was long-lived for more than 300 ms with high open probability (P(o), up to 0.8). This channel was permeable to Na(+) ions, but not to K(+), Ca(+), and Cl(-) ions, and was sensitively blocked by amiloride (200 nM). These characteristics and behaviors were quite similar to those of epithelial sodium channel (ENaC). RT-PCR and Western blot analyses confirmed that ENaC channel was endogenously expressed in PC12 cells. Taken together, a 30-pS ENaC-like channel was activated in response to acute hypoxia in PC12 cells. This is the first evidence of an acute hypoxia-activated Na(+) channel that can contribute to depolarization of the cell.

Lipid emulsion (LE) therapy has been used to reduce overdose of bupivacaine (BPV)-induced cardiotoxicity. The TWIK-related potassium channel-1 (TREK-1) is inhibited by BPV and activated by polyunsaturated fatty acids, which are the main component in LE. These pharmacological properties inspired us to investigate whether the TREK-1 channel is associated with cell viability of H9c2 cardiomyoblasts affected by BPV and LE. Consistent with previous studies, BPV-induced cell death was reduced by LE treatment. The reduction in the TREK-1 expression level by BPV was alleviated by LE. The BPV cytotoxicity highly decreased in TREK-1 overexpressed cells but was the opposite in TREK-1 knocked-down cells. TREK-1 channel activators and inhibitors increased and decreased cell viability, respectively. BPV-induced depolarization of the plasma and mitochondrial membrane potential and increase in intracellular Ca2+ level were blocked by LE treatment. BPV-induced depolarization of membrane potential was reduced in TREK-1 overexpressed cells, indicating that TREK-1 channels mediate setting the resting membrane potentials as a background K+ channel in H9c2 cells. These results show that TREK-1 activity is involved in the BPV cytotoxicity and the antagonistic effect of LE in H9c2 cells and suggest that TREK-1 could be a target for action of BPV and LE.

Earlier studies have demonstrated that the tandem pore domain weak inward rectifying K⁺ channel (TWIK)-related K⁺ (TREK)-1 channel is inhibited by antidepressants and is associated with major depression. However, little is known about the effect of mood stabilizers that are commonly used for treatment of bipolar disorder on TREK channels, members of the two-pore domain K⁺ (K2P) channel family. This study sought to investigate the effect of mood stabilizers on TREK-1 and TREK-2 channels. HEK-293A cells were transfected with human TREK-1 or TREK-2 DNA. The effect of mood stabilizers on TREK-1 and TREK-2 was studied using the patch clamp technique. Changes in TREK protein expression by mood stabilizers were studied in the HT-22 mouse hippocampal neuronal cells using western blot analysis. Lithium chloride (LiCl, 1 mM), gabapentin (100 μM), valproate (100 μM), and carbamazepine (100 μM) increased TREK-1 currents by 31 ± 14%, 25 ± 11%, 28 ± 12%, and 72 ± 12%, respectively, whereas they had no effect on TREK-2 channel activity. In addition, western blot analysis showed LiCl and carbamazepine slightly upregulated TREK-1 expression, but not TREK-2 in the HT-22 cells. These results suggest that TREK-1 could be a potential therapeutic target for treatment of bipolar disorders as well as depression, while TREK-2 is a target well suited for treatment of major depression.

IP3-induced Ca2+ release is the primary mechanism that is responsible for acetylcholine (ACh)-induced Ca2+ oscillation. However, other mechanisms remain to explain intracellular Ca2+ elevation. We here report that ACh induces Ca2+ influx via T-type Ca2+ channel by activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII), and the ACh-induced Ca2+ influx facilitates the generation of Ca2+ oscillation in the mouse ovulated oocytes (oocytes(MII)). ACh increased Ca2+ current by 50+/-21%, and produced Ca2+ oscillation. However, the currents and Ca2+ peaks were reduced in Ca2+ -free extracellular medium. ACh failed to activate Ca2+ current and to produce Ca2+ oscillation in oocytes pretreated with KN-93, a CaMKII inhibitor. KN-92, an inactive analogue of KN93, and PKC modulators could not prevent the effect of ACh. These results show that ACh increases T-type Ca2+ current by activation of CaMKII, independent of the PKC pathway, in the mouse oocytes.

Models created by the intraperitoneal injection of lipopolysaccharide (LPS) and D-galactosamine (D-GalN) have been widely used to study the pathogenesis of human acute liver failure (ALF) and drug development. Our previous study reported that oyster (Crassostrea gigas) hydrolysate (OH) had a hepatoprotective effect in LPS/D-GalN-injected mice. This study was performed to identify the hepatoprotective effect of the tyrosine-alanine (YA) peptide, the main component of OH, in a LPS/D-GalN-injected ALF mice model. We analyzed the effect of YA on previously known mechanisms of hepatocellular injury in the model. LPS/D-GalN-injected mice showed inflammatory, apoptotic, ferroptotic, and pyroptotic liver injury. The pre-administration of YA (10 mg/kg or 50 mg/kg) significantly reduced the liver damage factors. The hepatoprotective effect of YA was higher in the 50 mg/kg YA pre-administered group than in the 10 mg/kg YA pre-administered group. These results showed that YA had a hepatoprotective effect by reducing inflammation, apoptosis, ferroptosis, and pyroptosis in the LPS/D-GalN-injected ALF mouse model. We suggest that YA can be used as a functional peptide for the prevention of acute liver injury.