Oxidized Low-density Lipoprotein- and Lysophosphatidylcholine-induced Ca2+ Mobilization in Human Endothelial Cells

The effects of oxidized low-density lipoprotein (OxLDL) and its major lipid constituent lysophosphatidylcholine (LPC) on Ca²⁺ entry were investigated in cultured human umbilical endothelial cells (HUVECs) using fura-2 fluorescence and patch-clamp methods. OxLDL or LPC increased intracellular Ca²⁺ concentration ([Ca²⁺]_i), and the increase of [Ca²⁺]_i by OxLDL or by LPC was inhibited by La³⁺ or heparin. LPC failed to increase [Ca²⁺]_i in the presence of an antioxidant tempol. In addition, store-operated Ca²⁺ entry (SOC), which was evoked by intracellular Ca²⁺ store depletion in Ca²⁺-free solution using the sarcoplasmic reticulum Ca²⁺ pump blocker, 2, 5-di-t-butyl-1, 4-benzohydroquinone (BHQ), was further enhanced by OxLDL or by LPC. Increased SOC by OxLDL or by LPC was inhibited by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. In voltage-clamped cells, OxLDL or LPC increased [Ca²⁺]_i and simultaneously activated non-selective cation (NSC) currents. LPC-induced NSC currents were inhibited by 2-APB, La³⁺ or {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, and NSC currents were not activated by LPC in the presence of tempol. Furthermore, in voltage-clamped HUVECs, OxLDL enhanced SOC and evoked outward currents simultaneously. Clamping intracellular Ca²⁺ to 1 µM activated large-conductance Ca²⁺-activated K⁺ (BK_Ca) current spontaneously, and this activated BK_Ca current was further enhanced by OxLDL or by LPC. From these results, we concluded that OxLDL or its main component LPC activates Ca²⁺-permeable Ca²⁺-activated NSC current and BK_Ca current simultaneously, thereby increasing SOC.     

Lowering glucose level elevates [Ca2+]i in hypothalamic arcuate nucleus NPY neurons through P/Q-type Ca2+ channel activation and GSK3β inhibition

Aim:

To identify the mechanisms underlying the elevation of intracellular Ca²⁺ level ([Ca²⁺]_i) induced by lowering extracellular glucose in rat hypothalamic arcuate nucleus NPY neurons.

Methods:

Primary cultures of hypothalamic arcuate nucleus (ARC) neurons were prepared from Sprague-Dawley rats. NPY neurons were identified with immunocytochemical method. [Ca²⁺]_i was measured using fura-2 AM. Ca²⁺ current was recorded using whole-cell patch clamp recording. AMPK and GSK3β levels were measured using Western blot assay.

Results:

Lowering glucose level in the medium (from 10 to 1 mmol/L) induced a transient elevation of [Ca²⁺]_i in ARC neurons, but not in hippocampal and cortical neurons. The low-glucose induced elevation of [Ca²⁺]_i in ARC neurons depended on extracellular Ca²⁺, and was blocked by P/Q-type Ca²⁺channel blocker ω-agatoxin TK (100 nmol/L), but not by L-type Ca²⁺ channel blocker nifedipine (10 μmol/L) or N-type Ca²⁺channel blocker ω-conotoxin GVIA (300 nmol/L). Lowering glucose level increased the peak amplitude of high voltage-activated Ca²⁺ current in ARC neurons. The low-glucose induced elevation of [Ca²⁺]_i in ARC neurons was blocked by the AMPK inhibitor compound C (20 μmol/L), and enhanced by the GSK3β inhibitor LiCl (10 mmol/L). Moreover, lowering glucose level induced the phosphorylation of AMPK and GSK3β, which was inhibited by compound C (20 μmol/L).

Conclusion:

Lowering glucose level enhances the activity of P/Q type Ca²⁺channels and elevates [Ca²⁺]_i level in hypothalamic arcuate nucleus neurons via inhibition of GSK3β.     

Resveratrol protects rabbit ventricular myocytes against oxidative stress-induced arrhythmogenic activity and Ca2+ overload

Aim:

To investigate whether resveratrol suppressed oxidative stress-induced arrhythmogenic activity and Ca²⁺ overload in ventricular myocytes and to explore the underlying mechanisms.

Methods:

Hydrogen peroxide (H₂O₂, 200 μmol/L)) was used to induce oxidative stress in rabbit ventricular myocytes. Cell shortening and calcium transients were simultaneously recorded to detect arrhythmogenic activity and to measure intracellular Ca²⁺ ([Ca²⁺]_i). Ca²⁺/calmodulin-dependent protein kinases II (CaMKII) activity was measured using a CaMKII kit or Western blotting analysis. Voltage-activated Na⁺ and Ca²⁺ currents were examined using whole-cell recording in myocytes.

Results:

H₂O₂ markedly prolonged Ca²⁺ transient duration (CaTD), and induced early afterdepolarization (EAD)-like and delayed afterdepolarization (DAD)-like arrhythmogenic activity in myocytes paced at 0.16 Hz or 0.5 Hz. Application of resveratrol (30 or 50 μmol/L) dose-dependently suppressed H₂O₂-induced EAD-like arrhythmogenic activity and attenuated CaTD prolongation. Co-treatment with resveratrol (50 μmol/L) effectively prevented both EAD-like and DAD-like arrhythmogenic activity induced by H₂O₂. In addition, resveratrol markedly blunted H₂O₂-induced diastolic [Ca²⁺]_i accumulation and prevented the myocytes from developing hypercontracture. In whole-cell recording studies, H₂O₂ significantly enhanced the late Na⁺ current (I_Na,L) and L-type Ca²⁺ current (I_Ca,L) in myocytes, which were dramatically suppressed or prevented by resveratrol. Furthermore, H₂O₂-induced ROS production and CaMKII activation were significantly prevented by resveratrol.

Conclusion:

Resveratrol protects ventricular myocytes against oxidative stress-induced arrhythmogenic activity and Ca²⁺ overload through inhibition of I_Na,L/I_Ca,L, reduction of ROS generation, and prevention of CaMKII activation.     

Ketamine attenuates the Na+-dependent Ca2+ overload in rabbit ventricular myocytes in vitro by inhibiting late Na+ and L-type Ca2+ currents

Aim:

Intracellular Ca²⁺ ([Ca²⁺]_i) overload occurs in myocardial ischemia. An increase in the late sodium current (I_NaL) causes intracellular Na⁺ overload and subsequently [Ca²⁺]_i overload via the reverse-mode sodium-calcium exchanger (NCX). Thus, inhibition of INaL is a potential therapeutic target for cardiac diseases associated with [Ca²⁺]_i overload. The aim of this study was to investigate the effects of ketamine on Na⁺-dependent Ca²⁺ overload in ventricular myocytes in vitro.

Methods:

Ventricular myocytes were enzymatically isolated from hearts of rabbits. I_NaL, NCX current (I_NCX) and L-type Ca²⁺ current (I_CaL) were recorded using whole-cell patch-clamp technique. Myocyte shortening and [Ca²⁺]_i transients were measured simultaneously using a video-based edge detection and dual excitation fluorescence photomultiplier system.

Results:

Ketamine (20, 40, 80 μmol/L) inhibited I_NaL in a concentration-dependent manner. In the presence of sea anemone toxin II (ATX, 30 nmol/L), I_NaL was augmented by more than 3-fold, while ketamine concentration-dependently suppressed the ATX-augmented I_NaL. Ketamine (40 μmol/L) also significantly suppressed hypoxia or H₂O₂-induced enhancement of I_NaL. Furthermore, ketamine concentration-dependently attenuated ATX-induced enhancement of reverse-mode I_NCX. In addition, ketamine (40 μmol/L) inhibited I_CaL by 33.4%. In the presence of ATX (3 nmol/L), the rate and amplitude of cell shortening and relaxation, the diastolic [Ca²⁺]_i, and the rate and amplitude of [Ca²⁺]_i rise and decay were significantly increased, which were reverted to control levels by tetrodotoxin (TTX, 2 μmol/L) or by ketamine (40 μmol/L).

Conclusion:

Ketamine protects isolated rabbit ventricular myocytes against [Ca²⁺]_i overload by inhibiting I_NaL and I_CaL.     

Mechanisms of actions of hydrogen sulphide on rat distal colonic epithelium

BACKGROUND AND PURPOSE

The aim of this study was to clarify the mechanisms by which hydrogen sulphide (H₂S) affects ion secretion across rat distal colonic epithelium.

EXPERIMENTAL APPROACH

Changes in short-circuit current induced by the H₂S-donor, sodium hydrosulphide (NaHS; 10 mmol·L⁻¹), were measured in Ussing chambers after permeabilization of the apical membrane with nystatin. Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and Ca²⁺ in intracellular stores were measured with fluorescent dyes. Changes in mitochondrial membrane potential were estimated with rhodamine 123.

KEY RESULTS

NaHS had a biphasic effect on overall currents across the basolateral membrane: an initial inhibition followed by a secondary stimulation. Both a scilliroside-sensitive action on the Na⁺-K⁺-ATPase and modulation of glibenclamide-sensitive and tetrapentylammonium-sensitive (i.e. ATP-sensitive and Ca²⁺-dependent) basolateral K⁺ channels were involved in this action. Experiments with rhodamine 123 revealed that NaHS induced a hyperpolarization of the mitochondrial membrane. NaHS evoked a biphasic change in [Ca²⁺]_i, an initial decrease followed by a secondary increase, known to be mediated by the release of stored Ca²⁺. Initial falls in [Ca²⁺]_i were not mediated by a sequestration of Ca²⁺ in intracellular Ca²⁺ storing organelles, as the Mag-Fura-2 signal was unaffected by NaHS. Falls in [Ca²⁺]_i were inhibited by 2′,4′-dichlorobenzamil, an inhibitor of the Na⁺-Ca²⁺-exchanger, and attenuated in Na⁺-free buffer, suggesting a transient stimulation of Ca²⁺ outflow by this transporter, directly demonstrated by Mn²⁺ quenching experiments.

CONCLUSIONS AND IMPLICATIONS

ATP-sensitive and Ca²⁺-dependent basolateral K⁺ conductances, the basolateral Na⁺-K⁺-pump as well as Ca²⁺ transporters were involved in the action of H₂S in regulating colonic ion secretion.     

Functional comparison of the reverse mode of Na+/Ca2+ exchangers NCX1.1 and NCX1.5 expressed in CHO cells

Aim:

To investigate the reverse mode function of Na⁺/Ca²⁺ exchangers NCX1.1 and NCX1.5 expressed in CHO cells as well as their modulations by PKC and PKA.

Methods:

CHO-K1 cells were transfected with pcDNA3.1 (+) plasmid carrying cDNA of rat cardiac NCX1.1 and brain NCX1.5. The expression of NCX1.1 and NCX1.5 was examined using Western blot analysis. The intracellular Ca²⁺ level ([Ca²⁺]_i) was measured using Ca²⁺ imaging. Whole-cell NCX currents were recorded using patch-clamp technique. Reverse mode NCX activity was elicited by perfusion with Na⁺-free medium. Ca²⁺ paradox was induced by Ca²⁺-free EBSS medium, followed by Ca²⁺-containing solution (1.8 or 3.8 mmol/L CaCl₂).

Results:

The protein levels of NCX1.1 and NCX1.5 expressed in CHO cells had no significant difference. The reverse modes of NCX1.1 and NCX1.5 in CHO cells exhibited a transient increase of [Ca²⁺]_i, which was followed by a Ca²⁺ level plateau at higher external Ca²⁺ concentrations. In contrast, the wild type CHO cells showed a steady increase of [Ca²⁺]_i at higher external Ca²⁺ concentrations. The PKC activator PMA (0.3-10 μmol/L) and PKA activator 8-Br-cAMP (10-100 μmol/L) significantly enhanced the reverse mode activity of NCX1.1 and NCX1.5 in CHO cells. NCX1.1 was 2.4-fold more sensitive to PKC activation than NCX1.5, whereas the sensitivity of the two NCX isoforms to PKA activation had no difference. Both PKC- and PKA-enhanced NCX reverse mode activities in CHO cells were suppressed by NCX inhibitor KB-R7943 (30 μmol/L).

Conclusion:

Both NCX1.1 and NCX1.5 are functional in regulating and maintaining stable [Ca²⁺]_i in CHO cells and differentially regulated by PKA and PKC. The two NCX isoforms might be useful drug targets for heart and brain protection.     

U-73122, an aminosteroid phospholipase C inhibitor, may also block Ca2+ influx through phospholipase C-independent mechanism in neutrophil activation

1-[6-[[17a-3-Methoxyestra-1,3,5(10)-trien17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122) has been proven to be a useful tool in investigation of phospholipase C (PLC)-coupled signal transduction during cell activation. In the present studies, the inhibition by U-73122 of cytosolic free Ca²⁺ concentration ([Ca 2+]i) of neutrophils was investigated. U-73122 suppressed the [Ca²⁺]i elevation of neutrophils suspended in Ca²⁺-containing medium challenged by N-formyl-Met-Leu-Phe (fMLP), cyclopiazonic acid (CPA) and ionomycin. The concentrations of U-73122 required for inhibition of CPA- and ionomycin-induced changes with IC₅₀ values 4.06 ± 0.27 µM and 4.04 ± 0.44 µM, respectively, is almost 10-times that required for inhibition of the fMLP-induced response (IC₅₀ value 0.62 ± 0.04 µM) U-73122 also reduced the intracellular Ca²⁺ mobilization of neutrophils suspended in Ca ²⁺-free medium stimulated by fMLP and CPA, but not by ionomycin, with IC₅₀ values 0.52 ± 0.02 µM and 6.82 ± 0.74 µM, respectively. 1-[6-[[17f3-Methoxyestra-1,3,5(10)-trien-l7-yl]amino]hexyl]2,5-pyrrolidinedione (U-73343), a close analog of U-73122 that does not inhibit PLC activity, suppressed the [Ca²⁺]_i elevation of neutrophils challenged by fMLP in Ca²⁺-containing medium, but not in Ca²⁺-free medium, with IC₅₀ value 22.30 ± 1.61 µM. In Mn²⁺-quench studies, U-73122 suppressed the Mn²⁺ influx in CPA-activated neutrophils (IC₅₀ value was 7.16 ± 0.28 µM) as well as in resting neutrophils (IC₅₀ value was 6.72 ± 0.30 M). U-73343 also suppressed the Mn²⁺ influx in resting neutrophils in a concentration-dependent manner. These results suggest that the inhibitory effect of U-73122 on [Ca²⁺]i of activated neutrophils is attributed partly to the suppression of Ca²⁺ release from the intracellular Ca²⁺ stores through PLC inhibition, and partly to the blockade, especially at higher concentrations, of Ca²⁺ entry from the extracellular space through PLC-independent processes.     

Amiloride attenuates lipopolysaccharide-accelerated atherosclerosis via inhibition of NHE1-dependent endothelial cell apoptosis

Aim:

To investigate the effects of the potassium-sparing diuretic amiloride on endothelial cell apoptosis during lipopolysaccharide (LPS)-accelerated atherosclerosis.

Methods:

Human umbilical vein endothelial cells (HUVECs) were exposed to LPS (100 ng/mL) in the presence of drugs tested. The activity of Na⁺/H⁺ exchanger 1 (NHE1) and calpain, intracellular free Ca²⁺level ([Ca²⁺]_i), as well as the expression of apoptosis-related proteins in the cells were measured. For in vivo study, ApoE-deficient (ApoE^−/−) mice were fed high-fat diets with 0.5% (w/w) amiloride for 4 weeks and LPS (10 μg/mouse) infusion into caudal veins. Afterwards, atherosclerotic lesions, NHE1 activity and Bcl-2 expression in the aortic tissues were evaluated.

Results:

LPS treatment increased NHE1 activity and [Ca²⁺]_i in HUVECs in a time-dependent manner, which was associated with increased activity of the Ca²⁺-dependent protease calpain. Amiloride (1−10 μmol/L) significantly suppressed LPS-induced increases in NHE1 activity, [Ca²⁺]_i. and calpain activity. In the presence of the Ca²⁺ chelator BAPTA (0.5 mmol/L), LPS-induced increase of calpain activity was also abolished. In LPS-treated HUVECs, the expression of Bcl-2 protein was significantly decreased without altering its mRNA level. In the presence of amiloride (10 μmol/L) or the calpain inhibitor ZLLal (50 μmol/L), the down-regulation of Bcl-2 protein by LPS was blocked. LPS treatment did not alter the expression of Bax and Bak proteins in HUVECs. In the presence of amiloride, BAPTA or ZLLal, LPS-induced HUVEC apoptosis was significantly attenuated. In ApoE^−/− mice, administration of amiloride significantly suppressed LPS-accelerated atherosclerosis and LPS-induced increase of NHE1 activity, and reversed LPS-induced down-regulation of Bcl-2 expression.

Conclusion:

LPS stimulates NHE1 activity, increases [Ca²⁺]_i, and activates calpain, which leads to endothelial cell apoptosis related to decreased Bcl-2 expression. Amiloride inhibits NHE1 activity, thus attenuates LPS-accelerated atherosclerosis in mice.     

Differential calcium signalling by m2 and m3 muscarinic acetylcholine receptors in a single cell type

We have compared muscarinic acetylcholine receptor (mAChR) coupling to phospholipase C (PLC) and increases in cytoplasmic Ca²⁺ concentration [Ca²⁺]_i in human embryonic kidney (HEK) cells, stably expressing either the human m3 or m2 receptor subtype. In m3 mAChR-expressing cells, carbachol stimulated inositol phosphate (InsP) formation and increased [Ca²⁺]_i with EC₅₀ values of about 2 M and 30 nM, respectively. Maximal inositol 1,4,5-trisphosphate (InsP₃) production (about fourfold) was rapid (15 s) and stable for 2 min. Maximal increases in [Ca²⁺]_i were 300–350 nM and mainly, almost 90%, due to influx of extracellular Ca²⁺. The efficacy of pilocarpine for stimulating InsP andCa²⁺ responses was not significantly different from that of carbachol. All m3 mAChR-mediated responses were pertussis toxin (PTX)-insensitive. In m2 mAChR-expressing cells, carbachol stimulated InsP formation and increased [Ca²⁺]_i with EC₅₀ values of about 20 M and 7 M, respectively. Maximal InsP formation was only 10–15% of that observed in m3 mAChR-expressing cells, whereas maximal elevations of [Ca²⁺]_i were similar in both cell types. Formation of InsP₃ was rapid (15 s to 2 min) and about twofold above basal. In contrast to m3 mAChR activation, [Ca²⁺]_i increases induced by m2 mAChR activation were exclusively due to Ca²⁺ mobilization from intracellular stores.The efficacy of pilocarpine for stimulating InsP and Ca²⁺ responses was 50% and 20% of the efficacy of carbachol, respectively. PTX treatment did not affect m2 mAChR-induced PLC stimulation, but reduced the m2 mAChR-mediated increases in [Ca²⁺]_i to 50%. In conclusion, m3 and m2 mAChRs stably expressed in HEK cells can induce similar cellular responses; however, they do so by activating apparently distinct signalling pathways. While coupling of m2 mAChR to PLC occurs in a PTX-insensitive manner, coupling to mobilization of Ca²⁺ from intracellular stores is partly PTX-sensitive and this may occur at least partly independent of PLC activation.     

Independent [Ca2+]i increases and cell proliferation induced by the carcinogen safrole in human oral cancer cells

The effect of the carcinogen safrole on intracellular Ca²⁺ movement and cell proliferation has not been explored previously. The present study examined whether safrole could alter Ca²⁺ handling and growth in human oral cancer OC2 cells. Cytosolic free Ca²⁺ levels ([Ca²⁺]_i) in populations of cells were measured using fura-2 as a fluorescent Ca²⁺ probe. Safrole at a concentration of 325 M started to increase [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced by 40% by removing extracellular Ca²⁺, and was decreased by 39% by nifedipine but not by verapamil or diltiazem. In Ca²⁺-free medium, after pretreatment with 650 M safrole, 1 M thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor) barely induced a [Ca²⁺]_i rise; in contrast, addition of safrole after thapsigargin treatment induced a small [Ca²⁺]_i rise. Neither inhibition of phospholipase C with 2 M U73122 nor modulation of protein kinase C activity affected safrole-induced Ca²⁺ release. Overnight incubation with 1 M safrole did not alter cell proliferation, but incubation with 10–1000 M safrole increased cell proliferation by 60±10%. This increase was not reversed by pre-chelating Ca²⁺ with 10 M of the Ca²⁺ chelator BAPTA. Collectively, the data suggest that in human oral cancer cells, safrole induced a [Ca²⁺]_i rise by causing release of stored Ca²⁺ from the endoplasmic reticulum in a phospholipase C- and protein kinase C-independent fashion and by inducing Ca²⁺ influx via nifedipine-sensitive Ca²⁺ entry. Furthermore, safrole can enhance cell growth in a Ca²⁺-independent manner.     

Ca2+ entry following P2X receptor activation induces IP3 receptor-mediated Ca2+ release in myocytes from small renal arteries

BACKGROUND AND PURPOSE

P2X receptors mediate sympathetic control and autoregulation of the renal circulation triggering contraction of renal vascular smooth muscle cells (RVSMCs) via an elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Although it is well-appreciated that the myocyte Ca²⁺ signalling system is composed of microdomains, little is known about the structure of the [Ca²⁺]_i responses induced by P2X receptor stimulation in vascular myocytes.

EXPERIMENTAL APPROACHES

Using confocal microscopy, perforated-patch electrical recordings, immuno-/organelle-specific staining, flash photolysis and RT-PCR analysis we explored, at the subcellular level, the Ca²⁺ signalling system engaged in RVSMCs on stimulation of P2X receptors with the selective agonist αβ-methylene ATP (αβ-meATP).

KEY RESULTS

RT-PCR analysis of single RVSMCs showed the presence of genes encoding inositol 1,4,5-trisphosphate receptor type 1(IP₃R1) and ryanodine receptor type 2 (RyR2). The amplitude of the [Ca²⁺]_i transients depended on αβ-meATP concentration. Depolarization induced by 10 µmol·L⁻¹αβ-meATP triggered an abrupt Ca²⁺ release from sub-plasmalemmal (‘junctional’) sarcoplasmic reticulum enriched with IP₃Rs but poor in RyRs. Depletion of calcium stores, block of voltage-gated Ca²⁺ channels (VGCCs) or IP₃Rs suppressed the sub-plasmalemmal [Ca²⁺]_i upstroke significantly more than block of RyRs. The effect of calcium store depletion or IP₃R inhibition on the sub-plasmalemmal [Ca²⁺]_i upstroke was attenuated following block of VGCCs.

CONCLUSIONS AND IMPLICATIONS

Depolarization of RVSMCs following P2X receptor activation induces IP₃R-mediated Ca²⁺ release from sub-plasmalemmal (‘junctional’) sarcoplasmic reticulum, which is activated mainly by Ca²⁺ influx through VGCCs. This mechanism provides convergence of signalling pathways engaged in electromechanical and pharmacomechanical coupling in renal vascular myocytes.     

Quercetin induces insulin secretion by direct activation of L-type calcium channels in pancreatic beta cells

Background and Purpose

Quercetin is a natural polyphenolic flavonoid that displays anti-diabetic properties in vivo. Its mechanism of action on insulin-secreting beta cells is poorly documented. In this work, we have analysed the effects of quercetin both on insulin secretion and on the intracellular calcium concentration ([Ca²⁺]_i) in beta cells, in the absence of any co-stimulating factor.

Experimental Approach

Experiments were performed on both INS-1 cell line and rat isolated pancreatic islets. Insulin release was quantified by the homogeneous time-resolved fluorescence method. Variations in [Ca²⁺]_i were measured using the ratiometric fluorescent Ca²⁺ indicator Fura-2. Ca²⁺ channel currents were recorded with the whole-cell patch-clamp technique.

Key Results

Quercetin concentration-dependently increased insulin secretion and elevated [Ca²⁺]_i. These effects were not modified by the SERCA inhibitor thapsigargin (1 μmol·L⁻¹), but were nearly abolished by the L-type Ca²⁺ channel antagonist nifedipine (1 μmol·L⁻¹). Similar to the L-type Ca²⁺ channel agonist Bay K 8644, quercetin enhanced the L-type Ca²⁺ current by shifting its voltage-dependent activation towards negative potentials, leading to the increase in [Ca²⁺]_i and insulin secretion. The effects of quercetin were not inhibited in the presence of a maximally active concentration of Bay K 8644 (1 μmol·L⁻¹), with the two drugs having cumulative effects on [Ca²⁺]_i.

Conclusions and Implications

Taken together, our results show that quercetin stimulates insulin secretion by increasing Ca²⁺ influx through an interaction with L-type Ca²⁺ channels at a site different from that of Bay K 8644. These data contribute to a better understanding of quercetin''s mechanism of action on insulin secretion.     

Omega-3 fatty acids induce Ca2+ mobilization responses in human colon epithelial cell lines endogenously expressing FFA4

Aim:

Free fatty acid receptor 4 (FFA4; formerly known as GPR120) is the G protein-coupled receptor (GPCR) for omega-3 polyunsaturated fatty acids. FFA4 has been found to express in the small intestines and colons of mice and humans. In this study we investigate the effects of omega-3 polyunsaturated fatty acids on FFA4 in human colon epithelial cells in vitro.

Methods:

HCT116 and HT-29 human colon epithelial cell lines endogenously expressing FFA4 were used. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured in fura 2-AM-loaded cells with fluorescence spectrophotometry. RT-PCR and immunohistochemistry were used to detect FFA4.

Results:

Ten to 100 μmol/L of omega-3 polyunsaturated fatty acids α-linolenic acid (αLA) or eicosapentaenoic acid (EPA) induced dose-dependent [Ca²⁺]_i increase in HCT116 and HT-29 cells, whereas docosahexaenoic acid (DHA) had no effect. In addition, the omega-6 fatty acids linoleic acid and γ-linoleic acid also dose-dependently increase [Ca²⁺]_i, but the mono-unsaturated fatty acid oleic acid and saturated fatty acids such as stearic acid and palmitic acid had no effect. In HCT116 and HT-29 cells, the αLA-induced [Ca²⁺]_i increase was partially inhibited by pretreatment with EGTA, phospholipase C inhibitor edelfosine, cADPR inhibitors 8-bro-cADPR or DAB, and abolished by pretreatment with Ca²⁺ATPase inhibitor thapsigargin, but was not affected by G_i/o protein inhibitor PTX or IP₃R inhibitor 2-APB.

Conclusion:

Omega-3 and omega-6 long-chain polyunsaturated fatty acids (C18-20) induce Ca²⁺ mobilization responses in human colonic epithelial cells in vitro through activation of FFA4 and PTX-insensitive G_i/o protein, followed by Ca²⁺ release from thapsigargin-sensitive Ca²⁺ stores and Ca²⁺ influx across the plasma membrane.     

Ca2+ signals evoked by histamine H1 receptors are attenuated by activation of prostaglandin EP2 and EP4 receptors in human aortic smooth muscle cells

Background and Purpose

Histamine and prostaglandin E₂ (PGE₂), directly and via their effects on other cells, regulate the behaviour of vascular smooth muscle (VSM), but their effects on human VSM are incompletely resolved.

Experimental Approach

The effects of PGE₂ on histamine-evoked changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and adenylyl cyclase activity were measured in populations of cultured human aortic smooth muscle cells (ASMCs). Selective ligands of histamine and EP receptors were used to identify the receptors that mediate the responses.

Key Results

Histamine, via H₁ receptors, stimulates an increase in [Ca²⁺]_i that is entirely mediated by activation of inositol 1,4,5-trisphosphate receptors. Selective stimulation of EP₂ or EP₄ receptors attenuates histamine-evoked Ca²⁺ signals, but the effects of PGE₂ on both Ca²⁺ signals and AC activity are largely mediated by EP₂ receptors.

Conclusions and Implications

Two important inflammatory mediators, histamine via H₁ receptors and PGE₂ acting largely via EP₂ receptors, exert opposing effects on [Ca²⁺]_i in human ASMCs.     

The GPR55 agonist lysophosphatidylinositol relaxes rat mesenteric resistance artery and induces Ca2+ release in rat mesenteric artery endothelial cells

Background and Purpose

Lysophosphatidylinositol (LPI), a lipid signalling molecule, activates GPR55 and elevates intracellular Ca²⁺. Here, we examine the actions of LPI in the rat resistance mesenteric artery and Ca²⁺ responses in endothelial cells isolated from the artery.

Experimental Approach

Vascular responses were studied using wire myographs. Single-cell fluorescence imaging was performed using a MetaFluor system. Hypotensive effects of LPI were assessed using a Biopac system.

Key Results

In isolated arteries, LPI-induced vasorelaxation was concentration- and endothelium-dependent and inhibited by CID 16020046, a GPR55 antagonist. The CB₁ receptor antagonist AM 251 had no effect, whereas rimonabant and O-1918 significantly potentiated LPI responses. Vasorelaxation was reduced by charybdotoxin and iberiotoxin, alone or combined. LPI decreased systemic arterial pressure. GPR55 is expressed in rat mesenteric artery. LPI caused biphasic elevations of endothelial cell intracellular Ca²⁺. Pretreatment with thapsigargin or 2-aminoethoxydiphenyl borate abolished both phases. The PLC inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 attenuated the initial phase and enhanced the second phase, whereas the Rho-associated kinase inhibitor Y-27632 abolished the late phase but not the early phase.

Conclusions and Implications

LPI is an endothelium-dependent vasodilator in the rat small mesenteric artery and a hypotensive agent. The vascular response involves activation of Ca²⁺-sensitive K⁺ channels and is not mediated by CB₁ receptors, but unexpectedly enhanced by antagonists of the ‘endothelial anandamide’ receptor. In endothelial cells, LPI utilizes PLC-IP3 and perhaps ROCK-RhoA pathways to elevate intracellular Ca²⁺. Overall, these findings support an endothelial site of action for LPI and suggest a possible role for GPR55 in vasculature.     

Genetic polymorphisms and drug interactions modulating CYP2D6 and CYP3A activities have a major effect on oxycodone analgesic efficacy and safety

Background and purpose:

Thrombus formation is commonly associated with pulmonary arterial hypertension (PAH). Thrombin may thus play an important role in the pathogenesis and pathophysiology of PAH. Hence, we investigated the contractile effects of thrombin and its mechanism in pulmonary artery.

Experimental approach:

The cytosolic Ca²⁺ concentrations ([Ca²⁺]_i), 20 kDa myosin light chain (MLC20) phosphorylation and tension development were evaluated using the isolated porcine pulmonary artery.

Key results:

Thrombin induced a sustained contraction in endothelium-denuded strips obtained from different sites of a pulmonary artery, ranging from the main pulmonary artery to the intrapulmonary artery. In the presence of endothelium, thrombin induced a transient relaxation. The contractile effect of thrombin was abolished by either a protease inhibitor or a proteinase-activated receptor 1 (PAR₁) antagonist, while it was mimicked by PAR₁-activating peptide (PAR₁AP), but not PAR₄AP. The thrombin-induced contraction was associated with a small elevation of [Ca²⁺]_i and an increase in MLC20 phosphorylation. Thrombin and PAR₁AP induced a greater increase in tension for a given [Ca²⁺]_i elevation than that obtained with high K⁺-depolarization. They also induced a contraction at a fixed Ca²⁺ concentration in α-toxin-permeabilized preparations.

Conclusions and implications:

The present study revealed a unique property of the pulmonary artery. In contrast to normal arteries of the systemic circulation, thrombin induces a sustained contraction in the normal pulmonary artery, by activating PAR₁ and thereby increasing the sensitivity of the myofilament to Ca²⁺. This responsiveness of the pulmonary artery to thrombin may therefore contribute to the pathogenesis and pathophysiology of PAH.     

β-Asarone protects PC12 cells against OGD/R-induced injury via attenuating Beclin-1-dependent autophagy

Aim:

To explore the effects of β-asarone from Acorus Tatarinowii Schott on autophagy in an ischemic stroke model of PC12 cells.

Methods:

The ischemic stroke model of PC12 cells was made by OGD/R (2 h oxygen-glucose deprivation followed by 24 h reperfusion). Drug administration was started 1 h before OGD and last for 3 h. Then the cells were incubated in the drug-free and full culture medium under normoxic conditions for 24 h. After the treatments, Beclin-1, intracellular free calcium concentration ([Ca²⁺]_i) and mitochondrial membrane potential (MMP) were analyzed using flow cytometry. Cell viability was measured using MTT assay. Cell morphology was studied under inverted phase contrast microscope, and autophagosomes were observed under transmission electron microscope.

Results:

Pretreatment with β-asarone (20, 30, or 45 μg/mL) or the calcium channel antagonist nimodipine (10 μmol/L) significantly increased the cell viability and MMP, and decreased Beclin-1 expression and [Ca²⁺]_i in OGD/R-treated PC12 cells. Under inverted phase contrast microscope, pretreatment with β-asarone or nimodipine dramatically increase the number of cells and improved the cellular morphology. Autophagosomes were found in OGD/R-treated PC12 cells as well as in drug plus OGD/R-treated PC12 cells.

Conclusion:

β-Asarone protects PC12 cells against OGD/R-induced injury partly due to attenuating Beclin-1-dependent autophagy caused by decreasing [Ca²⁺]_i and increasing MMP.     

Acidic pH-activated Cl- Current and Intracellular Ca2+ Response in Human Keratinocytes

The layers of keratinocytes form an acid mantle on the surface of the skin. Herein, we investigated the effects of acidic pH on the membrane current and [Ca²⁺]_c of human primary keratinocytes from foreskins and human keratinocyte cell line (HaCaT). Acidic extracellular pH (pH_e≤ 5.5) activated outwardly rectifying Cl^- current (I_Cl,pH) with slow kinetics of voltage-dependent activation. I_Cl,pH was potently inhibited by an anion channel blocker 4,4''-diisothiocyanostilbene-2,2''-disulphonic acid (DIDS, 73.5% inhibition at 1 µM). I_Cl,pH became more sensitive to pH_e by raising temperature from 24℃ to 37℃. HaCaT cells also expressed Ca²⁺-activated Cl^- current (I_Cl,Ca), and the amplitude of I_Cl,Ca was increased by relatively weak acidic pH_e (7.0 and 6.8). Interestingly, the acidic pH_e (5.0) also induced a sharp increase in the intracellular [Ca²⁺] (Δ[Ca²⁺]_acid) of HaCaT cells. The Δ[Ca²⁺]_acid was independent of extracellular Ca²⁺, and was abolished by the pretreatment with PLC inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. In primary human keratinocytes, 5 out of 28 tested cells showed Δ[Ca²⁺]_acid. In summary, we found I_Cl,pH and Δ[Ca²⁺]_acid in human keratinocytes, and these ionic signals might have implication in pathophysiological responses and differentiation of epidermal keratinocytes.     

Inositol trisphosphate-dependent Ca2+ stores and mitochondria modulate slow wave activity arising from the smooth muscle cells of the guinea pig prostate gland

Background and purpose

Changes in smooth muscle tone of the prostate gland are involved in aetiology of symptomatic prostatic hyperplasia, however the control mechanisms of prostatic smooth muscle are not well understood. Here, we have examined the role of internal Ca²⁺ compartments in regulating slow wave activity in the guinea pig prostate.

Experimental approach

Standard intracellular membrane potential recording techniques were used.

Key results

The majority (89%) of impaled cells displayed ‘slow wave’ activity. Cyclopiazonic acid (10 µmol·L⁻¹) transiently depolarized (3–9 mV) the membrane potential of the prostatic stroma and transiently increased slow wave frequency. Thereafter, slow wave frequency slowly decreased over 20–30 min. Ryanodine transiently increased slow wave frequency, although after 30 min exposure slow wave frequency and time course returned to near control values. Caffeine (1 mmol·L⁻¹) reduced slow wave frequency, accompanied by membrane depolarization of about 8 mV. Blockade of inositol trisphosphate receptor (IP₃R)-mediated Ca²⁺ release with 2-aminoethoxy-diphenylborate (60 µmol·L⁻¹) or Xestospongin C (3 µmol·L⁻¹) or inhibiting phospholipase C and IP₃ formation using {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (5 µmol·L⁻¹) or neomycin (1 and 4 mmol·L⁻¹) reduced slow wave frequency, amplitude and duration. The mitochondrial uncouplers, p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (1–10 µmol·L⁻¹), carbonyl cyanide m-chlorophenylhydrazone (1–3 µmol·L⁻¹) or rotenone (10 µmol·L⁻¹), depolarized the membrane (8–10 mV) before abolishing electrical activity.

Conclusion and implications

These results suggest that slow wave activity was dependent on the cyclical release of Ca²⁺ from IP₃-controlled internal stores and mitochondria. This implies that intracellular compartments were essential in the initiation and/or maintenance of the regenerative contractile activity in the guinea pig prostate gland.