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Voltage-gated K + (Kv) channel opening repolarizes excitable cells by allowing K + efflux. Over the last two decades, multiple Kv functions in the nervous system have been found to be unrelated to or beyond the immediate control of excitability, such as shaping action potential contours or regulation of inter-spike frequency. These functions include neuronal exocytosis and neurite formation, neuronal cell death, regulation of astrocyte Ca 2+ , glial cell and glioma proliferation. Some of these functions have been shown to be independent of K + conduction, that is, they suggest the non-canonical functions of Kv channels. In this review, we focus on neuronal or glial plasmalemmal Kv channel functions which are unrelated to shaping action potentials or immediate control of excitability. Similar functions in other cell types will be discussed to some extent in appropriate contexts.

ATP depletion induced by inhibiting glycolysis or mitochondrial ATP production has been demonstrated to cause cancer cell death. Whether ATP depletion can enhance the efficacy and potency of anti-cancer effects of herbal compounds is so far unknown. We examined the enhancing effect of ATP depletion on anti-cancer actions of tetrandrine (TET) in human lung carcinoma A549 cells. A 24-h incubation of A549 cells with tetrandrine caused a concentration-dependent cytotoxic effect (LC50 = 66.1 μM). Co-incubation with 20 mM 2-deoxyglucose (2-DG, glycolysis inhibitor) caused only a very slight enhancement of tetrandrine cytotoxicity. By contrast, inhibiting mitochondrial ATP production with oligomycin (10 μM, ATP synthase inhibitor) and FCCP (30 μM, uncoupling agent) (thus, oligo-FCCP) on its own caused only slight cell cytotoxicity but strongly potentiated tetrandrine cytotoxicity (tetrandrine LC50 = 15.6 μM). The stronger enhancing effect of oligo-FCCP than 2-DG on TET toxicity did not result from more severe overall ATP depletion, since both treatments caused a similar ATP level suppression. Neither oligo-FCCP nor 2-DG synergized with tetrandrine in decreasing mitochondrial membrane potential. TET on its own triggered reactive oxygen species (ROS) production, and oligo-FCCP, but not 2-DG, potentiated TET in causing ROS production. Taken together, our results suggest that inhibiting ATP production from mitochondria, but not from glycolysis, appears to be a very effective means in augmenting TET-triggered ROS production and hence toxicity in A549 cells.

Substantiating evidence for the inflammation theory of depression is that interferon-alpha (IFN-α) induces clinical depression. Despite numerous researches on neurochemical and neuroendocrinological mechanisms from human and animal studies, the direct mechanisms of IFN-α at cellular levels are still lacking. In this study, we aimed to identify the cellular mechanisms for IFN-α-induced neuroinflammatory response with the murine BV-2 microglia cell line. IFN-α potently induced nitric oxide synthase (iNOS) and nitric oxide (NO) release and down-regulated haem oxygenase-1 (HO-1) expression, which could be dampened by Janus kinase 1 (JAK1) and c-Jun NH2-terminal kinase (JNK) inhibition, respectively. IFN-α activated JAK1, JNK, signal transducers and activators of transcription (STAT)1 and STAT3, but not extracellular signal-regulated kinases (ERK) and phosphoinositide 3 (PI3) kinase, signal pathways. The transfection with STAT1 and STAT3 siRNA also inhibited IFN-α-induced iNOS/NO expression and HO-1 down-regulation. The HO-1 activator, CoppIX, reversed iNOS/NO up-regulation and HO-1 down-regulation induced by IFN-α. On the other hand, a knockdown of HO-1 expression enhanced IFN-α-induced iNOS/NO expression. The effects of IFN-α-induced iNOS/NO up-regulation and HO-1 down-regulation in microglia are associated with JAK1/JNK/STAT1 and STAT3 signalling pathways. The different effects between IFN-α and IFN-γ on HO-1 regulation and ERK phosphorylation might provide a possible explanation of different risk in their induction of neuropsychiatric adverse effects in clinical and animal studies. The results from this study add the missing part of direct cellular mechanisms for IFN-α-induced depression.

Background This study investigated whether lipopolysaccharide (LPS) increase protease-activated receptor-2 (PAR-2) expression and enhance the association between PAR-2 expression and chemokine production in human vascular endothelial cells (ECs). Methods The morphology of ECs was observed through microphotography in cultured human umbilical vein ECs (EA. hy926 cells) treated with various LPS concentrations (0, 0.25, 0.5, 1, and 2 μg/mL) for 24 h, and cell viability was assessed using the MTT assay. Intracellular calcium imaging was performed to assess agonist (trypsin)-induced PAR-2 activity. Western blotting was used to explore the LPS-mediated signal transduction pathway and the expression of PAR-2 and adhesion molecule monocyte chemoattractant protein-1 (MCP-1) in ECs. Results Trypsin stimulation increased intracellular calcium release in ECs. The calcium influx was augmented in cells pretreated with a high LPS concentration (1 μg/mL). After 24 h treatment of LPS, no changes in ECs viability or morphology were observed. Western blotting revealed that LPS increased PAR-2 expression and enhanced trypsin-induced extracellular signal-regulated kinase (ERK)/p38 phosphorylation and MCP-1 secretion. However, pretreatment with selective ERK (PD98059), p38 mitogen-activated protein kinase (MAPK) (SB203580) inhibitors, and the selective PAR-2 antagonist (FSLLRY-NH2) blocked the effects of LPS-activated PAR-2 on MCP-1 secretion. Conclusions Our findings provide the first evidence that the bacterial endotoxin LPS potentiates calcium mobilization and ERK/p38 MAPK pathway activation and leads to the secretion of the pro-inflammatory chemokine MCP-1 by inducing PAR-2 expression and its associated activity in vascular ECs. Therefore, PAR-2 exerts vascular inflammatory effects and plays an important role in bacterial infection-induced pathological responses.

Accumulating evidence suggests that the pathophysiology of depression might be associated with neuroinflammation, which could be attenuated by pharmacological treatment for depression. Omega-3 polyunsaturated fatty acids (PUFAs) are anti-inflammatory and exert antidepressant effects. The aim of this study was to identify the molecular mechanisms through which docosahexaenoic acid (DHA), the main omega-3 PUFA in the brain, modulates oxidative reactions and inflammatory cytokine production in microglial and neuronal cells. The results of this study showed that DHA reduced expressions of tumor necrosis factor- α , interleukin-6, nitric oxide synthase, and cyclo-oxygenase-2, induced by interferon- γ , and induced upregulation of heme oxygenase-1 (HO-1) in BV-2 microglia. The inhibitory effect of DHA on nitric oxide production was abolished by HO-1 inhibitor zinc protoporphyrin IX. In addition, DHA caused AKT and ERK activation in a time-dependent manner, and the DHA-induced HO-1 upregulation could be attenuated by PI-3 kinase/AKT and MEK/ERK inhibitors. DHA also increased IKK α / β phosphorylation, I κ B α phosphorylation, and I κ B α degradation, whereas both nuclear factor- κ B and I κ B protease inhibitors could inhibit DHA-induced HO-1 expressions. The other major n-3 PUFA, eicosapentaenoic acid, showed similar effects of DHA on inflammation and HO-1 in repeated key experiments. In connecting with inflammation hypothesis of depression and clinical studies supporting the antidepressant effects of omega-3 PUFAs, this study provides a novel implication of the antidepressant mechanisms of DHA.

The expression of matrix metalloproteinase-13 (MMP-13) has been shown to be elevated in some pathophysiological conditions and is involved in the degradation of extracellular matrix in astrocytes. In current study, the function of MMP-13 was further investigated. The conditioned medium (CM) collected from activated microglia increased interleukin (IL)-18 production and enhanced MMP-13 expression in astrocytes. Furthermore, treatment with recombinant IL-18 increased MMP-13 protein and mRNA levels in astrocytes. Recombinant IL-18 stimulation also increased the enzymatic activity of MMP-13 and the migratory activity of astrocytes, while administration of MMP-13 or pan-MMP inhibitors antagonized IL-18-induced migratory activity of astrocytes. In addition, administration of recombinant IL-18 to astrocytes led to the phosphorylation of JNK, Akt, or PKCδ, and treatment of astrocytes with JNK, PI3 kinase/Akt, or PKCδ inhibitors significantly decreased the IL-18-induced migratory activity. Taken together, the results suggest that IL-18-induced MMP-13 expression in astrocytes is regulated by JNK, PI3 kinase/Akt, and PKCδ signaling pathways. These findings also indicate that IL-18 is an important regulator leading to MMP-13 expression and cell migration in astrocytes.

Ca?·+-sensing receptors (CaSR) are G-protein coupled receptors activated by elevated concentrations of extracellular Ca?·. Cinacalcet, a positive allosteric CaSR modulator, is used as a calcimimetic to inhibit parathyroid hormone release and thus lower serum Ca?· in hypercalcemic patients. There is a recent trend of repurposing cinacalcet in multiple applications such as anti-cancer actions, treatment of diarrhea and prevention of kidney cyst formation. In this study we investigated whether cinacalcet inhibited proliferation of endothelial cells (EC). Inhibition of EC proliferation offers an anti-angiogenesis mechanism in suppressing tumor growth. Cinacalcet at >18 ?? caused mitochondrial membrane depolarization, suppressed proliferation and induced apoptosis in mouse bEND.3 EC. In 2 mM Ca?·-containing bath solution, cinacalcet (>18 uM) caused an initial rise in [Ca?·]; followed by fura-2 leakage. Similar results were obtained in Ca'··-free or 4 mM Ca\"·-containing bath solution. Cinacalcet-elicited Ca2+ signal was unaffected by NPS 2143, a negative allosteric modulator of CaSR. Cinacalcet also increased Ni?· leakage and trypan blue uptake into cells. Cinacalcet-induced membrane leakiness and cytotoxicity did not appear to be related to membrane fluidity changes. These data suggest cinacalcet, in a manner independent of CaSR stimulation and membrane fluidity perturbation, caused membrane leakiness, eventually leading to inhibition of EC proliferation and EC death.

Trans-cinnamaldehyde (TCA), an essential oil in cinnamon powder, may have beneficial effects as a treatment for stroke which is the second leading cause of death worldwide. Post-ischemic inflammation induces neuronal cell damage after stroke, and activation of microglia, in particular, has been thought as the main contributor of proinflammatory and neurotoxic factors. The purpose of this study was to investigate the neuroprotective effects of TCA in an animal model of ischemia/reperfusion (I/R)-induced brain injury and the neuroprotective mechanism was verified in LPS-induced inflammation of BV-2 microglial cells. Our results showed that TCA (10–30 mg/kg, p.o.) significantly reduced the infarction area, neurological deficit score and decreased iNOS and COX-2 protein expression level in I/R-induced injury brain tissue. It inhibited 0.5 µg/ml LPS-induced NO production in BV-2 microglial cells without affecting cell viability, reduced protein expression of iNOS and COX-2, and attenuated inhibition of p53 protein. TCA also suppressed the effects of LPS-induced nuclear translocation of NF-κB p65 and p50 and increased cytosolic IκBα. It also reduced LPS-induced mRNA expression of iNOS, COX-2, and TNFα. We concluded that TCA has a potential neuroprotective effect to against the ischemic stroke, which may be via the inhibition of neuroinflammation through attenuating iNOS, COX-2 expression and NF-κB signaling pathway.

Transgenic Mouse Overexpressing Syntaxin-1A as a Diabetes Model Patrick P.L. Lam 1 2 , Yuk-Man Leung 1 2 , Laura Sheu 1 2 , James Ellis 3 , Robert G. Tsushima 1 2 , Lucy R. Osborne 1 4 and Herbert Y. Gaisano 1 2 1 Department of Medicine, University of Toronto, Toronto, Canada 2 Department of Physiology, University of Toronto, Toronto, Canada 3 Program in Developmental Biology, Sick Kids Hospital, Toronto, Canada 4 Department of Molecular and Medical Genetics, University of Toronto, Toronto, Canada Address correspondence and reprint requests to Herbert Y. Gaisano, MD, University of Toronto, Room 7226, Medical Science Building, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8. E-mail: herbert.gaisano{at}utoronto.ca . Or Lucy R. Osborne, PhD, University of Toronto, Room 7238, Medical Science Building, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8. E-mail: lucy.osborne{at}utoronto.ca Abstract Soluble N -ethylmaleimide–sensitive factor (NSF) attachment protein receptor (SNARE) protein syntaxin-1A (STX-1A) plays a role not only in exocytosis, but also binds and regulates Ca 2+ and K + (voltage-gated K + and ATP-sensitive K + channels) to influence the sequence of events leading to secretion. Islet levels of STX-1A and cognate SNARE proteins are reduced in type 2 diabetic rodents, suggesting their role in dysregulated insulin secretion contributing to the abnormal glucose homeostasis. We investigated the specific role of STX-1A in pancreatic β-cells by generating transgenic mice, which express a moderately increased level (∼30% higher) of STX-1A in pancreatic islets (hereafter called STX-1A mice). The STX-1A mice displayed fasting hyperglycemia and a more sustained elevation of plasma glucose levels after an intraperitoneal glucose tolerance test, with correspondingly reduced plasma insulin levels. Surprisingly, β-cells from the STX-1A male mice also exhibited abnormal insulin tolerance. To unequivocally determine the β-cell secretory defects, we used single-cell analyses of exocytosis by patch clamp membrane capacitance measurements and ion channel recordings. Depolarization-evoked membrane capacitance increases were reduced in the STX-1A mouse islet β-cells. The STX-1A mouse also exhibited reduced currents through the Ca 2+ channels but little change in the voltage-gated K + channel or ATP-sensitive K + channel. These results suggest that fluctuation of islet STX-1A levels in diabetes could influence the pathological and differential regulation of β-cell ion channels and the exocytotic machinery, collectively contributing to the impaired insulin secretion. Cm, membrane capacitance GST, glutathione S-transferase IPGTT, intraperitoneal glucose tolerance test KATP channel, ATP-sensitive K+ channel Kv channel, voltage-gated potassium channel NSF, N-ethylmaleimide–sensitive factor SNAP-25, synaptosome-associated protein of 25 kDa SNARE, soluble NSF attachment protein receptor STX-1A, syntaxin-1A TEA, tetraethylammonium VAMP-2, vesicle-associated membrane protein 2 WBS, Williams-Beuren syndrome Footnotes P.P.L.L. and Y.-M.L. contributed equally to this work. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted June 7, 2005. Received November 23, 2004. DIABETES

Ca2+-sensing receptors (CaSR) are G protein-coupled receptors which are activated by a rise in extracellular Ca2+. CaSR activation has been known to inhibit parathyroid hormone release and stimulate calcitonin release from parathyroid glands and thyroid parafollicular C cells, respectively. The roles of CaSR in other cell types including endothelial cells (EC) are much less understood. In this work, we demonstrated protein and functional expression of CaSR in mouse cerebral EC (bEND.3). Unexpectedly, CaSR response (high Ca2+-elicited cytosolic [Ca2+] elevation) was unaffected by edelfosine or U73122 but strongly suppressed by SK&F 96365, ruthenium red, and 2-aminoethoxydiphenyl borate (2-APB), suggesting involvement of TRPV and TRPC channels but not Gq-phospholipase C. Acute application of NPS2143, a negative allosteric modulator of CaSR, suppressed CaSR response. However, a 40-min NPS2143 pre-treatment surprisingly enhanced CaSR response. After 4–24 h of application, this enhancement faded away and suppression of CaSR response was observed again. Similar results were obtained when La3+ and Sr2+ were used as CaSR agonists. The transient NPS 2143 enhancement effect was abolished by SB203580, a p38 inhibitor. Consistently, NPS 2143 triggered a transient p38 activation. Taken together, results suggest that in bEND.3 cells, NPS 2143 caused acute suppression of CaSR response, but then elicited a transient enhancement of CaSR response in a p38-dependent manner. NPS 2143 effects on CaSR in bEND.3 cells therefore depended on drug exposure time. These findings warrant cautious use of this agent as a CaSR modulator and potential cardiovascular drug.