Ca2+-sensing receptor-TRP channel-mediated Ca2+ signaling: Functional diversity and pharmacological complexity.

The Ca2+-sensing receptor (CaSR) is a G-protein-coupled receptor activated by elevated concentrations of extracellular Ca2+, and was initially known for its regulation of parathyroid hormone (PTH) release. Ubiquitous expression of CaSR in different tissues and organs was later noted and CaSR participation in various physiological functions was demonstrated. Accumulating evidence has suggested that CaSR functionally interacts with transient receptor potential (TRP) channels, which are mostly non-selective cation channels involved in sensing temperature, pain and stress. This review describes the interactions of CaSR with TRP channels in diverse cell types to trigger a variety of biological responses. CaSR has been known to interact with different types of G proteins. Possible involvements of G proteins, other signaling and scaffolding protein intermediates in CaSR-TRP interaction are discussed. In addition, an attempt will be made to extend the current understanding of biased agonism of CaSR.

Suppression of Ca2+ oscillations by SERCA inhibition in human alveolar type 2 A549 cells: rescue by ochratoxin A but not CDN1163.

AIMS: Lung type 2 alveolar cells, by secreting surfactant to lower surface tension, contribute to enhance lung compliance. Stretching, as a result of lung expansion, triggers type 1 alveolar cell to release ATP, which in turn stimulates Ca2+-dependent surfactant secretion by neighboring type 2 cells. In this report, we studied ATP-triggered Ca2+ signaling in human alveolar type 2 A549 cells. MAIN METHODS: Ca2+ signaling was examined using microfluorimetric measurement with fura-2 as fluorescent dye. KEY FINDINGS: Ca2+ oscillations triggered by ATP relied on inositol 1,4,5-trisphosphate-induced Ca2+ release and store-operated Ca2+ entry. Pathological conditions such as influenza virus infection and diabetes reportedly inhibit sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA). We found that a very mild inhibition of SERCA by cyclopiazonic acid (CPA) sufficed to decrease Ca2+ oscillation frequency and the percentage of cells exhibiting Ca2+ oscillations. Ochratoxin A (OTA), an activator of SERCA, could prevent the suppressive effects by CPA. Inhibition of SERCA by hydrogen peroxide also suppressed Ca2+ oscillations. Interestingly, hydrogen peroxide-induced inhibition was prevented by OTA but aggravated by CDN1163, an allosteric activator of SERCA. CDN1163 also had an untoward effect of releasing intracellular Ca2+. SIGNIFICANCE: Different modes of activation of SERCA may determine the outcome of rescue of Ca2+ oscillations in case of SERCA inhibition in alveolar type 2 cells.

Drug Repurposing: The Mechanisms and Signaling Pathways of Anti-Cancer Effects of Anesthetics.

Cancer is one of the leading causes of death worldwide. There are only limited treatment strategies that can be applied to treat cancer, including surgical resection, chemotherapy, and radiotherapy, but these have only limited effectiveness. Developing a new drug for cancer therapy is protracted, costly, and inefficient. Recently, drug repurposing has become a rising research field to provide new meaning for an old drug. By searching a drug repurposing database ReDO_DB, a brief list of anesthetic/sedative drugs, such as haloperidol, ketamine, lidocaine, midazolam, propofol, and valproic acid, are shown to possess anti-cancer properties. Therefore, in the current review, we will provide a general overview of the anti-cancer mechanisms of these anesthetic/sedative drugs and explore the potential underlying signaling pathways and clinical application of these drugs applied individually or in combination with other anti-cancer agents.

Sensitivity of Ca2+-sensing receptor-transient receptor potential-mediated Ca2+ influx to extracellular acidity in bEND.3 endothelial cells.

Ca2+-sensing receptors (CaSRs) are G protein-coupled receptors activated by elevated concentrations of extracellular Ca2+. In our previous works, we showed protein and functional expression of CaSR in mouse cerebral endothelial cell (EC) (bEND.3); the CaSR response (high Ca2+-elicited cytosolic [Ca2+] elevation) was unaffected by suppression of phospholipase C but in part involved Ca2+ influx through transient receptor potential V1 (TRPV1) channels. In this work, we investigated if extracellular acidity affected CaSR-mediated Ca2+ influx triggered by high (3 mM) Ca2+ (CaSR agonist), 3 mM spermine (CaSR agonist), and 10 mM cinacalcet (positive allosteric modulator of CaSR). Extracellular acidosis (pH 6.8 and pH 6.0) strongly suppressed cytosolic [Ca2+] elevation triggered by high Ca2+, spermine, and cinacalcet; acidosis also inhibited Mn2+ influx stimulated by high Ca2+ and cinacalcet. Purinoceptor-triggered Ca2+ response, however, was not suppressed by acidosis. Extracellular acidity also did not affect membrane potential, suggesting suppressed CaSR-mediated Ca2+ influx in acidity did not result from the reduced electrical driving force for Ca2+. Our results suggest Ca2+ influx through a putative CaSR-TRP complex in bEND.3 EC was sensitive to extracellular pH.

Anticancer Effects of Midazolam on Lung and Breast Cancers by Inhibiting Cell Proliferation and Epithelial-Mesenchymal Transition.

Despite improvements in cancer treatments resulting in higher survival rates, the proliferation and metastasis of tumors still raise new questions in cancer therapy. Therefore, new drugs and strategies are still needed. Midazolam (MDZ) is a common sedative drug acting through the γ-aminobutyric acid receptor in the central nervous system and also binds to the peripheral benzodiazepine receptor (PBR) in peripheral tissues. Previous studies have shown that MDZ inhibits cancer cell proliferation but increases cancer cell apoptosis through different mechanisms. In this study, we investigated the possible anticancer mechanisms of MDZ on different cancer cell types. MDZ inhibited transforming growth factor ß (TGF-ß)-induced cancer cell proliferation of both A549 and MCF-7 cells. MDZ also inhibited TGF-ß-induced cell migration, invasion, epithelial-mesenchymal-transition, and Smad phosphorylation in both cancer cell lines. Inhibition of PBR by PK11195 rescued the MDZ-inhibited cell proliferation, suggesting that MDZ worked through PBR to inhibit TGF-ß pathway. Furthermore, MDZ inhibited proliferation, migration, invasion and levels of mesenchymal proteins in MDA-MD-231 triple-negative breast cancer cells. Together, MDZ inhibits cancer cell proliferation both in epithelial and mesenchymal types and EMT, indicating an important role for MDZ as a candidate to treat lung and breast cancers.

A basal level of γ-linolenic acid depletes Ca2+ stores and induces endoplasmic reticulum and oxidative stresses to cause death of breast cancer BT-474 cells.

Gamma-linolenic acid (GLA), a natural fatty acid obtained from oils of various vegetables and seeds, has been demonstrated as an anticancer agent. In this work, we investigated the anticancer effects of GLA on breast cancer BT-474 cells. GLA at 30 µM, a concentration reportedly within the range of circulating concentrations in clinical studies, caused apoptotic cell death. GLA caused an elevation in mitochondrial Ca2+ level and a decrease in mitochondrial membrane potential. GLA treatment depleted cyclopiazonic acid (CPA)-sensitive Ca2+ store and triggered substantial Ca2+ influx. Intracellular Ca2+ release triggered by GLA was suppressed by 3 µM xestospongin C (XeC, IP3 receptor-channel blocker) and 100 µM ryanodine (ryanodine receptor-channel blocker), suggesting that the Ca2+ release was via IP3 receptor-channel and ryanodine receptor-channel. Increased expressions of p-eIF2α and CHOP were observed in GLA-treated cells, suggesting GLA-treated cells had increased expressions of p-eIF2α and CHOP, which suggest endoplasmic reticulum (ER) stress. In addition, GLA elicited increased production of reactive oxygen species. Taken together, our results suggest a basal level of GLA induced apoptotic cell death by causing Ca2+ overload, mitochondrial dysfunction, Ca2+ store depletion, ER stress, and oxidative stress. This is the first report to show that GLA caused Ca2+ store depletion and ER stress. GLA-induced Ca2+ store depletion resulted from opening of IP3 receptor-channel and ryanodine receptor-channel.

Perturbation of Ca2+ stores and store-operated Ca2+ influx by lidocaine in neuronal N2A and NG108-15 cells.

In this report we examined the effects of lidocaine on Ca2+ homeostasis of neuronal cells using microfluorimetric measurement of cytosolic Ca2+ with fura 2 as probe. In mouse neuroblastoma N2A cells, 10 mM lidocaine caused Ca2+ release from the cyclopiazonic acid (CPA)-dischargeable pool and abolished ATP-triggered Ca2+ release. Lidocaine-triggered Ca2+ release was not affected by xestospongin C (XeC), an inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor. N2A cells did not have functional ryanodine receptors (RYR) (absence of caffeine response) and we used differentiated NG108-15 cells (presence of caffeine response) for further experiments. Caffeine-triggered Ca2+ release was unaffected by a brief lidocaine exposure, but was eliminated after a prolonged treatment of lidocaine, suggesting lidocaine abolished caffeine action possibly not by interfering caffeine binding but via Ca2+ store depletion. Lidocaine-elicited Ca2+ release was unaffected by XeC or a high concentration of ryanodine, suggesting Ca2+ release was not via IP3R or RYR. Lidocaine did not affect nigericin-dischargeable lysosomal Ca2+ stores. Lastly, we observed that lidocaine suppressed CPA-induced store-operated Ca2+ influx in both N2A cells and differentiated NG108-15 cells. Our results suggest two novel actions of lidocaine in neuronal cells, namely, depletion of Ca2+ store (via an IP3R- and RYR-independent manner) and suppression of store-operated Ca2+ influx.

Aqueous extract of Arctium lappa L. root (burdock) enhances chondrogenesis in human bone marrow-derived mesenchymal stem cells.

BACKGROUND: Arctium lappa L. root (burdock root) has long been recommended for the treatment of different diseases in traditional Chinese medicine. Burdock root possesses anti-oxidative, anti-inflammatory, anti-cancer, and anti-microbial activities. The aim of the study was to elucidate whether aqueous extract of burdock root regulates mesenchymal stem cell proliferation and differentiation. METHODS: Human bone marrow-derived mesenchymal stem cells in 2D high density culture and in 3D micromass pellets were treated with chondrogenic induction medium and chondral basal medium in the absence or presence of aqueous extract of burdock root. The chondrogenic differentiation was accessed by staining glucosaminoglycans, immunostaining SOX9 and type II collagen and immuonblotting of SOX9, aggrecan and type II collagen. RESULTS: Treatment of aqueous extract of burdock root increased the cell proliferation of hMSCs. It did not have significant effect on osteogenic and adipogenic differentiation, but significantly enhanced chondrogenic induction medium-induced chondrogenesis. The increment was dose dependent, as examined by staining glucosaminoglycans, SOX9, and type II collagen and immunobloting of SOX9, aggrecan and type II collagen in 2D and 3D cultures. In the presence of supplemental materials, burdock root aqueous extract showed equivalent chondrogenic induction capability to that of TGF-ß. CONCLUSIONS: The results demonstrate that aqueous extract of Arctium lappa L. root promotes chondrogenic medium-induced chondrogenic differentiation. The aqueous extract of burdock root can even be used alone to stimulate chondrogenic differentiation. The study suggests that the aqueous extract of burdock root can be used as an alternative strategy for treatment purposes.

Polyamine stimulation perturbs intracellular Ca2+ homeostasis and decreases viability of breast cancer BT474 cells.

Intracellular polyamines such as spermine and spermidine are essential to cell growth in normal and especially in cancer cells. However, whether extracellular polyamines affect cancer cell survival is unknown. We therefore examined the actions of extracellular polyamines on breast cancer BT474 cells. Our data showed that spermine, spermidine, and putrescine decreased cell viability by apoptosis. These polyamines also elicited Ca2+ signals, but the latter were unlikely triggered via Ca2+-sensing receptor (CaSR) as BT474 cells have been demonstrated previously to lack CaSR expression. Spermine-elicited Ca2+ response composed of both Ca2+ release and Ca2+ influx. Spermine caused a complete discharge of the cyclopiazonic acid (CPA)-sensitive Ca2+ pool and, expectedly, endoplasmic reticulum (ER) stress. The Ca2+ influx pore opened by spermine was Mn2+-impermeable, distinct from the CPA-triggered store-operated Ca2+ channel, which was Mn2+-permeable. Spermine cytotoxic effects were not due to oxidative stress, as spermine did not trigger reactive oxygen species formation. Our results therefore suggest that spermine acted on a putative polyamine receptor in BT474 cells, causing cytotoxicity by Ca2+ overload, Ca2+ store depletion, and ER stress.

ARC 118925XX stimulates cation influx in bEND.3 endothelial cells.

In a previous publication when we studied the purinergic receptor with which ATP interacted in mouse brain bEND.3 endothelial cells, we observed addition of 3 µm ARC 118925XX (ARC; selective P2Y2 antagonist) strongly suppressed ATP-triggered Ca2+ release, suggesting the response was mediated via P2Y2 receptors. We here report ARC unexpectedly promoted substantial Ca2+ influx even when ATP-triggered Ca2+ release was largely inhibited. Since this large Ca2+ influx may have important pharmacological significance, we proceeded to investigate its mechanism. ARC did not trigger intracellular Ca2+ release thus suggesting Ca2+ influx triggered by ARC was not store-operated. ARC-triggered Ca2+ influx could be blocked by 1 mm Ni2+ , a general Ca2+ channel blocker, but not by SK&F 96365, a nonselective TRP channel blocker. Unexpectedly, ARC promoted influx of Na+ and La3+ , but not Mn2+ . This is a surprising finding, since Mn2+ is conventionally used as a Ca2+ surrogate ion (as it permeates Ca2+ channel), and La3+ is classically used as a potent Ca2+ channel antagonist. Electrophysiological examination showed ARC did not stimulate any cation currents. Therefore, ARC opened, rather than a cation channel pore, an unidentified Ca2+ influx pathway which was Na+ - and La3+ -permeable but Mn2+ -impermeable.

Etomidate Suppresses Invasion and Migration of Human A549 Lung Adenocarcinoma Cells.

BACKGROUND/AIM: Etomidate, an intravenous anesthetic, has been shown to have anticancer effects, including induction of cell-cycle arrest and apoptosis. However, to our knowledge, there are no reports about the anti-metastasis effects of etomidate on A549 human lung adenocarcinoma cells. MATERIALS AND METHODS: The cell viability, cell adhesion, gelatin zymography assay, transwell migration and invasion assay, and western blotting analysis were used to investigate the effects of etomidate on A549 cells. RESULTS: In our study, etomidate showed low cytotoxicity, inhibited cell adhesion, and suppressed the migration and invasion in A549 cells. The activity of matrix metallopeptidase 2 (MMP2) was reduced by 48 h treatment of etomidate. Results of western blotting analysis indicated that etomidate down-regulated the expression of protein kinase C, MMP7, MMP1, MMP9, and p-p-38, but up-regulated that of RAS, phosphoinositide 3-kinase, and phosphor-extracellular-signal related kinase after 24 and 48 h treatment, in A549 cells. CONCLUSION: Etomidate suppressed the migration and invasion of lung adenocarcinoma A549 cells via inhibiting the expression of MMP1, MMP2, MMP7 and MMP9, and provides potential therapeutic targets for lung cancer treatment.

Midazolam inhibits chondrogenesis via peripheral benzodiazepine receptor in human mesenchymal stem cells.

Midazolam, a benzodiazepine derivative, is widely used for sedation and surgery. However, previous studies have demonstrated that Midazolam is associated with increased risks of congenital malformations, such as dwarfism, when used during early pregnancy. Recent studies have also demonstrated that Midazolam suppresses osteogenesis of mesenchymal stem cells (MSCs). Given that hypertrophic chondrocytes can differentiate into osteoblast and osteocytes and contribute to endochondral bone formation, the effect of Midazolam on chondrogenesis remains unclear. In this study, we applied a human MSC line, the KP cell, to serve as an in vitro model to study the effect of Midazolam on chondrogenesis. We first successfully established an in vitro chondrogenic model in a micromass culture or a 2D high-density culture performed with TGF-ß-driven chondrogenic induction medium. Treatment of the Midazolam dose-dependently inhibited chondrogenesis, examined using Alcian blue-stained glycosaminoglycans and the expression of chondrogenic markers, such as SOX9 and type II collagen. Inhibition of Midazolam by peripheral benzodiazepine receptor (PBR) antagonist PK11195 or small interfering RNA rescued the inhibitory effects of Midazolam on chondrogenesis. In addition, Midazolam suppressed transforming growth factor-ß-induced Smad3 phosphorylation, and this inhibitory effect could be rescued using PBR antagonist PK11195. This study provides a possible explanation for Midazolam-induced congenital malformations of the musculoskeletal system through PBR.

Eicosapentaenoic acid triggers Ca2+ release and Ca2+ influx in mouse cerebral cortex endothelial bEND.3 cells.

Eicosapentaenoic acid (EPA), an omega-3 fatty acid abundant in fish oil, protects endothelial cells (EC) from lipotoxicity and triggers EC NO release. The latter is related to an elevation of cytosolic Ca2+. Although EPA has been shown to cause human EC cytosolic Ca2+ elevation, the mechanism is unclear. Microfluorimetric imaging was used here to measure free cytosolic Ca2+ concentration. EPA was shown to cause intracellular Ca2+ release in mouse cerebral cortex endothelial bEND.3 cells; interestingly, the EPA-sensitive intracellular Ca2+ pool(s) appeared to encompass and was larger than the Ca2+ pool mobilized by sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibition by cyclopiazonic acid. EPA also opened a Ca2+ influx pathway pharmacologically distinct from store-operated Ca2+ influx. Surprisingly, EPA-triggered Ca2+ influx was Ni2+-insensitive; and EPA did not trigger Mn2+ influx. Further, EPA-triggered Ca2+ influx did not involve Na+-Ca2+ exchangers. Thus, our results suggest EPA triggered unusual mechanisms of Ca2+ release and Ca2+ influx in EC.

Perturbation of Akt Signaling, Mitochondrial Potential, and ADP/ATP Ratio in Acidosis-Challenged Rat Cortical Astrocytes.

Cells switch to anaerobic glycolysis when there is a lack of oxygen during brain ischemia. Extracellular pH thus drops and such acidosis causes neuronal cell death. The fate of astrocytes, mechanical, and functional partners of neurons, in acidosis is less studied. In this report, we investigated the signaling in acidosis-challenged rat cortical astrocytes and whether these signals were related to mitochondrial dysfunction and cell death. Exposure to acidic pH (6.8, 6.0) caused Ca2+ release and influx, p38 MAPK activation, and Akt inhibition. Mitochondrial membrane potential was hyperpolarized after astrocytes were exposed to acidic pH as soon as 1 h and lasted for 24 h. Such mitochondrial hyperpolarization was prevented by SC79 (an Akt activator) but not by SB203580 (a p38 inhibitor) nor by cytosolic Ca2+ chelation by BAPTA, suggesting that only the perturbation in Akt signaling was causally related to mitochondrial hyperpolarization. SC79, SB203580, and BAPTA did not prevent acidic pH-induced cell death. Acidic pH suppressed ROS production, thus ruling out the role of ROS in cytotoxicity. Interestingly, pH 6.8 caused an increase in ADP/ATP ratio and apoptosis; pH 6.0 caused a further increase in ADP/ATP ratio and necrosis. Therefore, astrocyte cell death in acidosis did not result from mitochondrial potential collapse; in case of acidosis at pH 6.0, necrosis might partly result from mitochondrial hyperpolarization and subsequent suppressed ATP production. J. Cell. Biochem. 118: 1108-1117, 2017. © 2016 Wiley Periodicals, Inc.

Benzyl isothiocyanate alters the gene expression with cell cycle regulation and cell death in human brain glioblastoma GBM 8401 cells.

Glioblastoma multiforme (GBM) is a highly malignant devastating brain tumor in adults. Benzyl isothiocyanate (BITC) is one of the isothiocyanates that have been shown to induce human cancer cell apoptosis and cell cycle arrest. Herein, the effect of BITC on cell viability and apoptotic cell death and the genetic levels of human brain glioblastoma GBM 8401 cells in vitro were investigated. We found that BITC induced cell morphological changes, decreased cell viability and the induction of cell apoptosis in GBM 8401 cells was time-dependent. cDNA microarray was used to examine the effects of BITC on GBM 8401 cells and we found that numerous genes associated with cell death and cell cycle regulation in GBM 8401 cells were altered after BITC treatment. The results show that expression of 317 genes was upregulated, and two genes were associated with DNA damage, the DNA-damage-inducible transcript 3 (DDIT3) was increased 3.66-fold and the growth arrest and DNA-damage-inducible α (GADD45A) was increased 2.34-fold. We also found that expression of 182 genes was downregulated and two genes were associated with receptor for cell responses to stimuli, the EGF containing fibulin-like extracellular matrix protein 1 (EFEMP1) was inhibited 2.01-fold and the TNF receptor-associated protein 1 (TRAP1) was inhibited 2.08-fold. BITC inhibited seven mitochondria ribosomal genes, the mitochondrial ribosomal protein; tumor protein D52 (MRPS28) was inhibited 2.06-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein L23 (MRPL23) decreased 2.08-fold, the mitochondria ribosomal protein S2 (MRPS2) decreased 2.07-fold, the mitochondria ribosomal protein S12 (MRPS12) decreased 2.08-fold, the mitochondria ribosomal protein L12 (MRPL12) decreased 2.25-fold and the mitochondria ribosomal protein S34 (MRPS34) was decreased 2.30-fold in GBM 8401 cells. These changes of gene expression can provide the effects of BITC on the genetic level and are potential biomarkers for glioblastoma therapy.

Inhibition of voltage-gated Na+ channels by hinokiol in neuronal cells.

BACKGROUND: Hinokiol is a naturally occurring diterpenoid compound isolated from plants such as Taiwania cryptomerioides. Anti-oxidation, anti-cancer, and anti-inflammation effects of this compound have been reported. It is not yet known if hinokiol affects neurons or neuronal ion channel activities. We reported here that hinokiol inhibited voltage-gated Na(+) channels (VGSC) in neuronal cells and we characterized the mechanisms of block. METHODS: The effects of hinokiol on Na(+) channels were examined using the voltage-clamp (whole-cell mode) technique. RESULTS: VGSC was blocked by hinokiol in a concentration-dependent and state-dependent manner in neuroblastoma N2A cells: IC(50) are 11.3 and 37.4µM in holding potentials of -70 and -100 mV, respectively. In the presence of hinokiol there was a 13-mV left shift in steady-state inactivation curves; however, activation gating was not altered. VGSC inhibition by hinokiol did not require channel opening and was thus considered to be closed-channel block. In the presence of hinokiol, since the degree of block did not enhance with stimulation frequency, block by hinokiol thus did not exhibit use-dependence. Recovery from channel inactivation was not significantly affected in the presence of hinokiol. In addition, hinokiol also inhibited VGSC of differentiated neuronal NG108-15 cells and rat hippocampal CA1 neurons. CONCLUSION: Our results therefore suggest hinokiol inhibited VGSC in a closed-channel block manner and such inhibition involved intensification of channel inactivation.

Role of voltage-gated K(+) channels in regulating Ca(2+) entry in rat cortical astrocytes.

Astrocytes have multiple functions such as provision of nourishment and mechanical support to the nervous system, helping to clear extracellular metabolites of neurons and modulating synaptic transmission by releasing gliotransmitters. In excitable cells, voltage-gated K(+) (Kv) channels serve to repolarize during action potentials. Astrocytes are considered non-excitable cells since they are not able to generate action potentials. There is an abundant expression of various Kv channels in astrocytes but the functions of these Kv channels remain unclear. We examined whether these astrocyte Kv channels regulate astrocyte "excitability" in the form of cytosolic Ca(2+) signaling. Electrophysiological examination revealed that neonatal rat cortical astrocytes possessed both delayed rectifier type and A-type Kv channels. Pharmacological blockade of both delayed rectifier Kv channels by TEA and A-type Kv channels by quinidine significantly suppressed store-operated Ca(2+) influx; however, TEA alone or quinidine alone did not suffice to cause such suppression. TEA and quinidine together dramatically enhanced current injection-triggered membrane potential overshoot (depolarization); either drug alone caused much smaller enhancements. Taken together, the results suggest both delayed rectifier and A-type Kv channels regulate astrocyte Ca(2+) signaling via controlling membrane potential.

Different cellular responses of dexmedetomidine at infected site and peripheral blood of emdotoxemic BALB/c mice.

Various sedative agents, including dexmedetomidine (dex), induce immunosuppression, and enhance infection progression. However, there was no information on how anesthetic affects local and systemic cellular immune function. We conducted this study to examine the impact of dex on the differentiation and function of immune cells at site of inflammation and in peripheral blood during endotoxemia of mice. In BALB/c mice with and without endotoxemia, we evaluated the influence of two dosages of 5 and 50 mcg/kg/h intravenous dex on immune cells: including number of T cells (CD3), B cells (CD19), natural killer cells (CD8a), monocytes (CD11b), and macrophages (Mac-3) in peripheral blood, the activities of macrophages in peripheral blood and in peritoneal lavage, and proliferation of B and T cells and of natural killer cells activity in the spleen. Endotoxemia increased the number of CD3 T cells, CD 19 B cells and macrophages in the peripheral blood, augmented macrophage activity in the peritoneum, and increased T cell proliferation and natural killer cell activity in the spleen. Further administration of 5 mcg/kg/h dex attenuated systemic increase in number of T cells, B cells, and macrophages during endotoxemia and 50 mcg/kg/h dex significantly attenuated the increase in activity of macrophages in the peripheral blood during endotoxemia. In the peritoneum, however, 5 mcg/kg/h dex preserved and 50 mcg/kg/h dexmedetomidine enhanced the activity of macrophages during endotoxemia. Increased in proliferation of T cells in spleen during endotoxemia was attenuated by both doses of dex. Last, 50 mcg/kg/h dex enhanced natural killer cells activity during endotoxemia. While preserving the effects of endotoxemia on macrophage's activity in the infection site and natural killer cell's activity in the spleen, dex decreased systemic fulminant immune reaction in endotoxemia, by attenuating the augmented response in the number of T cells, B cells and macrophages, activity of macrophages in the peripheral blood, and proliferation of T cells in spleen during endotoxemia.

Effects of midazolam on ion currents and membrane potential in differentiated motor neuron-like NSC-34 and NG108-15 cells.

Midazolam (MDL) was known to act through stimulation of benzodiazepine receptors (GABA). Whether midazolam affects ion currents and membrane potential in neurons remains largely unclear. Electrophysiological studies of midazolam actions were performed in differentiated motor neuron-like (NSC-34 and NG108-15) cells. Midazolam suppressed the amplitude of delayed rectifier K(+) current (IK(DR)) in a time- and concentration-dependent manner with an IC50 value of 10.4 µM. Addition of midazolam was noted to enhance the rate of IK(DR) inactivation. On the basis of minimal binding scheme, midazolam-induced block of IK(DR) was quantitatively provided with a dissociation constant of 9.8 µM. Recovery of IK(DR) from inactivation in the presence of midazolam was fitted by a single exponential. midazolam had no effect on M-type or erg-mediated K(+) current in these cells. Midazaolam (30 µM) suppressed the peak amplitude of voltage-gated Na(+) current (INa) with no change in the current-voltage relationships of this current. Inactivation kinetics of INa remained unaltered in the presence of this agent. In current-clamp configuration, midazolam (30 µM) prolonged the duration of action potentials (APs) and reduce AP amplitude. Similarly, in differentiated NG108-15 cells, the exposure to midazolam also suppressed IK(DR) with a concomitant increase in current inactivation. Midazolam can act as an open-channel blocker of delayed-rectifier K(+) channels in these cells. The synergistic blocking effects on IK(DR) and INa may contribute to the underlying mechanisms through which midazolam affects neuronal function in vivo.

Suppression of voltage-gated Na(+) channels and neuronal excitability by imperatorin.

Imperatorin is a naturally occurring furocoumarin compound isolated from plants such as Angelica archangelica and Cnidium monnieri. It has multiple pharmacological effects including anticonvulsant effects. Here we determined the effects of imperatorin on voltage-gated Na(+) channels (VGSC) using whole-cell patch clamp techniques in differentiated neuronal NG108-15 cells. We showed that imperatorin inhibited VGSC; such inhibition did not show state-dependence. Imperatorin caused a left shift in the steady-state inactivation curve without affecting activation gating. The inhibition of VGSC by imperatorin displayed a mild frequency-dependence. Imperatorin was also shown to inhibit VGSC and action potential amplitude without affecting voltage-gated K(+) channels in rat hippocampal CA1 neurons. In conclusion, our results suggest that imperatorin dampens neuronal excitability by inhibiting VGSC.