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.     

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.     

Hydrogen peroxide mobilizes Ca2+ through two distinct mechanisms in rat hepatocytes

Aim:

Hydrogen peroxide (H₂O₂) is produced during liver transplantation. Ischemia/reperfusion induces oxidation and causes intracellular Ca²⁺ overload, which harms liver cells. Our goal was to determine the precise mechanisms of these processes.

Methods:

Hepatocytes were extracted from rats. Intracellular Ca²⁺ concentrations ([Ca²⁺]_i), inner mitochondrial membrane potentials and NAD(P)H levels were measured using fluorescence imaging. Phospholipase C (PLC) activity was detected using exogenous PIP₂. ATP concentrations were measured using the luciferin-luciferase method. Patch-clamp recordings were performed to evaluate membrane currents.

Results:

H₂O₂ increased intracellular Ca²⁺ concentrations ([Ca²⁺]_i) across two kinetic phases. A low concentration (400 μmol/L) of H₂O₂ induced a sustained elevation of [Ca²⁺]_i that was reversed by removing extracellular Ca²⁺. H₂O₂ increased membrane currents consistent with intracellular ATP concentrations. The non-selective ATP-sensitive cation channel blocker amiloride inhibited H₂O₂-induced membrane current increases and [Ca²⁺]_i elevation. A high concentration (1 mmol/L) of H₂O₂ induced an additional transient elevation of [Ca²⁺]_i, which was abolished by the specific PLC blocker {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 but was not eliminated by removal of extracellular Ca²⁺. PLC activity was increased by 1 mmol/L H₂O₂ but not by 400 μmol/L H₂O₂.

Conclusion:

H₂O₂ mobilizes Ca²⁺ through two distinct mechanisms. In one, 400 μmol/L H₂O₂-induced sustained [Ca²⁺]_i elevation is mediated via a Ca²⁺ influx mechanism, under which H₂O₂ impairs mitochondrial function via oxidative stress, reduces intracellular ATP production, and in turn opens ATP-sensitive, non-specific cation channels, leading to Ca²⁺ influx. In contrast, 1 mmol/L H₂O₂-induced transient elevation of [Ca²⁺]_i is mediated via activation of the PLC signaling pathway and subsequently, by mobilization of Ca²⁺ from intracellular Ca²⁺ stores.     

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.     

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.     

Eleutheroside B,a selective late sodium current inhibitor,suppresses atrial fibrillation induced by sea anemone toxin Ⅱ in rabbit hearts

Eleutheroside B(EB)is the main active constituent derived from the Chinese herb Acanthopanax senticosus(AS)that has been reported to possess cardioprotective effects.In this study we investigated the effects of EB on cardiac electrophysiology and its suppression on atrial fibrillation(AF).Whole-cell recording was conducted in isolated rabbit atrial myocytes.The intracellular calcium([Ca²⁺]i)concentration was measured using calcium indicator Fura-2/AM fluorescence.Monophasic action potential(MAP)and electrocardiogram(ECG)synchronous recordings were conducted in Langendorff-perfused rabbit hearts using ECG signal sampling and analysis system.We showed that EB dose-dependently inhibited late sodium current(INaL),transient sodium current(INaT),and sea anemone toxin II(ATX II)-increased INaL with IC50 values of 167,1582,and 181μM,respectively.On the other hand,EB(800μM)did not affect L-type calcium current(ICaL),inward rectifier potassium channel current(IK),and action potential duration(APD).Furthermore,EB(300μM)markedly decreased ATX II-prolonged the APD at 90%repolarization(APD90)and eliminated ATX II-induced early afterdepolarizations(EADs),delayed afterdepolarizations(DADs),and triggered activities(TAs).Moreover,EB(200μM)significantly suppressed ATX II-induced Na+-dependent[Ca2+]i overload in atrial myocytes.In the Langendorff-perfused rabbit hearts,application of EB(200μM)or TTX(2μM)substantially decreased ATX II-induced incidences of atrial fibrillation(AF),ventricular fibrillation(VF),and heart death.These results suggest that augmented INaL alone is sufficient to induce AF,and EB exerts anti-AF actions mainly via blocking INaL,which put forward the basis of pharmacology for new clinical application of EB.     

The TRPC channel blocker SKF 96365 inhibits glioblastoma cell growth by enhancing reverse mode of the Na+/Ca2+ exchanger and increasing intracellular Ca2+

BACKGROUND AND PURPOSE

SKF 96365 is well known for its suppressing effect on human glioblastoma growth by inhibiting pre-activated transient receptor potential canonical (TRPC) channels and Ca²⁺ influx. The effect of SKF 96363 on glioblastoma cells, however, may be multifaceted and this possibility has been largely ignored.

EXPERIMENTAL APPROACH

The effects of SKF 96365 on cell cycle and cell viability of cultured human glioblastoma cells were characterized. Western blot, Ca²⁺ imaging and patch clamp recordings were used to delineate cell death mechanisms. siRNA gene knockdown provided additional evidence.

KEY RESULTS

SKF 96365 repressed glioblastoma cell growth via increasing intracellular Ca²⁺ ([Ca²⁺]_i) irrespective of whether TRPC channels were blocked or not. The effect of SKF 96365 primarily resulted from enhanced reverse operation of the Na⁺/Ca²⁺ exchanger (NCX) with an EC₅₀ of 9.79 μM. SKF 96365 arrested the glioblastoma cells in the S and G₂ phases and activated p38-MAPK and JNK, which were all prevented by the Ca²⁺ chelator BAPTA-AM or EGTA. The expression of NCX in glioblastoma cells was significantly higher than in normal human astrocytes. Knockdown of the NCX1 isoforms diminished the effect of SKF 96365 on glioblastoma cells.

CONCLUSIONS AND IMPLICATIONS

At the same concentration, SKF 96365 blocks TRPC channels and enhances the reverse mode of the NCX causing [Ca²⁺]_i accumulation and cytotoxicity. This finding suggests an alternative pharmacological mechanism of SKF 96365. It also indicates that modulation of the NCX is an effective method to disrupt Ca²⁺ homeostasis and suppress human glioblastoma cells.     

Doxorubicin-induced vasomotion and [Ca^2＋]i elevation n vascular smooth muscle cells from C57BL/6 mice

Aim:

To explore the action of doxorubicin on vascular smooth muscle cells.

Methods:

Isometric tension of denuded or intact thoracic aortic vessels was recorded and [Ca²⁺]_i in isolated aortic smooth muscle cells was measured by using Fluo-3.

Results:

Doxorubicin induced phasic and tonic contractions in denuded vessels and increased levels of [Ca²⁺]_i in single muscle cells. Treatment with 10 μmol/L ryanodine had no effect on basal tension, but it did abolish doxorubicin-induced phasic contraction. Treatment with 10 mmol/L caffeine induced a transient phasic contraction only, and the effect was not significantly altered by ryanodine, the omission of extracellular Ca²⁺ or both. Phenylephrine induced rhythmic contraction (RC) in intact vessels. Treatment with 100 μmol/L doxorubicin enhanced RC amplitude, but 1 mmol/L doxorubicin abolished RC, with an increase in maximal tension. Caffeine at 100 μmol/L increased the frequency of the RC only. In the presence of 100 μmol/L caffeine, however, 100 μmol/L doxorubicin abolished the RC and decreased its maximal tension. Treatment with 10 μmol/L ryanodine abolished the RC, with an increase in the maximal tension. In Ca²⁺-free solution, doxorubicin induced a transient [Ca²⁺]_i increase that could be abolished by ryanodine pretreatment in single muscle cells. The doxorubicin-induced increase in [Ca²⁺]_i was suppressed by nifedipine and potentiated by ryanodine and charybdotoxin.

Conclusion:

Doxorubicin not only releases Ca²⁺ from the sarcoplasmic reticulum but also promotes the entry of extracellular Ca²⁺ into vascular smooth muscle cells.     

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.     

Magnesium lithospermate B extracted from Salvia miltiorrhiza elevats intracellular Ca2＋ level in SHSY5Y cells

Aim:

To examine if magnesium lithospermate B (MLB), a potent inhibitor of Na⁺/K⁺-ATPase, leads to the elevation of intracellular Ca²⁺ level as observed in cells treated with cardiac glycosides.

Methods:

Viability of SH-SY5Y neuroblastoma cells treated with various concentrations of ouabain or MLB was measured. Intracellular Ca²⁺ levels were visualized using Fluo4-AM (fluorescent dye) when cells were treated with ouabain or MLB in the presence or absence of KB-R7943 (Na⁺/Ca²⁺ exchanger inhibitor) and 2-APB (IP₃ receptor antagonist). Molecular modeling was conducted for the docking of ouabain or MLB to Na⁺/K⁺-ATPase. Changes of cell body and dendrite morphology were monitored under a microscope.

Results:

severe toxicity was observed in cells treated with ouabain of concentration higher than 1 μmol/L for 24 h while no apparent toxicity was observed in those treated with MLB. Intracellular Ca²⁺ levels were substantially elevated by MLB (1 μmol/L) and ouabain (1 μmol/L) in similar patterns, and significantly reduced in the presence of KB-R7943 (10 μmol/L) or 2-APB (100 μmol/L). Equivalent interaction with the binding cavity of Na⁺/K⁺-ATPase was simulated for ouabain and MLB by forming five hydrogen bonds, respectively. Treatment of ouabain (1 μmol/L), but not MLB (1 μmol/L), induced dendritic shrink of SH-SY5Y cells.

Conclusion:

Comparable to ouabain, MLB leads to the elevation of intracellular Ca²⁺ level presumably via the same mechanism by inhibiting Na⁺/K⁺-ATPase. The elevated Ca²⁺ levels seem to be supplied by Ca²⁺ influx through the reversed mode of the Na⁺/Ca²⁺ exchanger and intracellular release from endoplasmic reticulum.     

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.     

N-arachidonoyl glycine suppresses Na+/Ca2+ exchanger-mediated Ca2+ entry into endothelial cells and activates BKCa channels independently of GPCRs

Background and Purpose

N-arachidonoyl glycine (NAGly) is a lipoamino acid with vasorelaxant properties. We aimed to explore the mechanisms of NAGly''s action on unstimulated and agonist-stimulated endothelial cells.

Experimental Approach

The effects of NAGly on endothelial electrical signalling were studied in combination with vascular reactivity.

Key Results

In EA.hy926 cells, the sustained hyperpolarization to histamine was inhibited by the non-selective Na⁺/Ca²⁺ exchanger (NCX) inhibitor bepridil and by an inhibitor of reversed mode NCX, KB-R7943. In cells dialysed with Cs⁺-based Na⁺-containing solution, the outwardly rectifying current with typical characteristics of NCX was augmented following histamine exposure, further increased upon external Na⁺ withdrawal and inhibited by bepridil. NAGly (0.3–30 μM) suppressed NCX currents in a URB597- and guanosine 5′-O-(2-thiodiphosphate) (GDPβS)-insensitive manner, [Ca²⁺]_i elevation evoked by Na⁺ removal and the hyperpolarization to histamine. In rat aorta, NAGly opposed the endothelial hyperpolarization and relaxation response to ACh. In unstimulated EA.hy926 cells, NAGly potentiated the whole-cell current attributable to large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in a GDPβS-insensitive, paxilline-sensitive manner and produced a sustained hyperpolarization. In cell-free inside-out patches, NAGly stimulated single BK_Ca channel activity.

Conclusion and Implications

Our data showed that NCX is a Ca²⁺ entry pathway in endothelial cells and that NAGly is a potent G-protein-independent modulator of endothelial electrical signalling and has a dual effect on endothelial electrical responses. In agonist pre-stimulated cells, NAGly opposes hyperpolarization and relaxation via inhibition of NCX-mediated Ca²⁺ entry, while in unstimulated cells, it promotes hyperpolarization via receptor-independent activation of BK_Ca channels.     

Congo red modulates ACh-induced Ca2+ oscillations in single pancreatic acinar cells of mice

Aim:

Congo red, a secondary diazo dye, is usually used as an indicator for the presence of amyloid fibrils. Recent studies show that congo red exerts neuroprotective effects in a variety of models of neurodegenerative diseases. However, its pharmacological profile remains unknown. In this study, we investigated the effects of congo red on ACh-induced Ca²⁺ oscillations in mouse pancreatic acinar cells in vitro.

Methods:

Acutely dissociated pancreatic acinar cells of mice were prepared. A U-tube drug application system was used to deliver drugs into the bath. Intracellular Ca²⁺ oscillations were monitored by whole-cell recording of Ca²⁺-activated Cl⁻ currents and by using confocal Ca²⁺ imaging. For intracellular drug application, the drug was added in pipette solution and diffused into cell after the whole-cell configuration was established.

Results:

Bath application of ACh (10 nmol/L) induced typical Ca²⁺ oscillations in dissociated pancreatic acinar cells. Addition of congo red (1, 10, 100 μmol/L) dose-dependently enhanced Ach-induced Ca²⁺ oscillations, but congo red alone did not induce any detectable response. Furthermore, this enhancement depended on the concentrations of ACh: congo red markedly enhanced the Ca²⁺ oscillations induced by ACh (10–30 nmol/L), but did not alter the Ca²⁺ oscillations induced by ACh (100–10000 nmol/L). Congo red also enhanced the Ca²⁺ oscillations induced by bath application of IP₃ (30 μmol/L). Intracellular application of congo red failed to alter ACh-induced Ca²⁺ oscillations.

Conclusion:

Congo red significantly modulates intracellular Ca²⁺ signaling in pancreatic acinar cells, and this pharmacological effect should be fully considered when developing congo red as a novel therapeutic drug.     

Ca2+ sparks promote myogenic tone in retinal arterioles

Background and Purpose

Ca²⁺ imaging reveals subcellular Ca²⁺ sparks and global Ca²⁺ waves/oscillations in vascular smooth muscle. It is well established that Ca²⁺ sparks can relax arteries, but we have previously reported that sparks can summate to generate Ca²⁺ waves/oscillations in unpressurized retinal arterioles, leading to constriction. We have extended these studies to test the functional significance of Ca²⁺ sparks in the generation of myogenic tone in pressurized arterioles.

Experimental Approach

Isolated retinal arterioles (25–40 μm external diameter) were pressurized to 70 mmHg, leading to active constriction. Ca²⁺ signals were imaged from arteriolar smooth muscle in the same vessels using Fluo4 and confocal laser microscopy.

Key Results

Tone development was associated with an increased frequency of Ca²⁺ sparks and oscillations. Vasomotion was observed in 40% of arterioles and was associated with synchronization of Ca²⁺ oscillations, quantifiable as an increased cross-correlation coefficient. Inhibition of Ca²⁺ sparks with ryanodine, tetracaine, cyclopiazonic acid or nimodipine, or following removal of extracellular Ca²⁺, resulted in arteriolar relaxation. Cyclopiazonic acid-induced dilatation was associated with decreased Ca²⁺ sparks and oscillations but with a sustained rise in the mean global cytoplasmic [Ca²⁺] ([Ca²⁺]_c), as measured using Fura2 and microfluorimetry.

Conclusions and Implications

This study provides direct evidence that Ca²⁺ sparks can play an excitatory role in pressurized arterioles, promoting myogenic tone. This contrasts with the generally accepted model in which sparks promote relaxation of vascular smooth muscle. Changes in vessel tone in the presence of cyclopiazonic acid correlated more closely with changes in spark and oscillation frequency than global [Ca²⁺]_c, underlining the importance of frequency-modulated signalling in vascular smooth muscle.     

Forskolin Changes the Relationship between Cytosolic Ca2+ and Contraction in Guinea Pig Ileum

This study was designed to clarify the mechanism of the inhibitory effect of forskolin on contraction, cytosolic Ca²⁺ level ([Ca²⁺]_i), and Ca²⁺ sensitivity in guinea pig ileum. Forskolin (0.1 nM~10 µM) inhibited high K⁺ (25 mM and 40 mM)- or histamine (3 µM)-evoked contractions in a concentration-dependent manner. Histamine-evoked contractions were more sensitive to forskolin than high K⁺-evoked contractions. Spontaneous changes in [Ca²⁺]_i and contractions were inhibited by forskolin (1 µM) without changing the resting [Ca²⁺]_i. Forskoln (10 µM) inhibited muscle tension more strongly than [Ca²⁺]_i stimulated by high K⁺, and thus shifted the [Ca²⁺]_i-tension relationship to the lower-right. In histamine-stimulated contractions, forskolin (1 µM) inhibited both [Ca²⁺]_i and muscle tension without changing the [Ca²⁺]_i-tension relationship. In α-toxin-permeabilized tissues, forskolin (10 µM) inhibited the 0.3 µM Ca²⁺-evoked contractions in the presence of 0.1 mM GTP, but showed no effect on the Ca²⁺-tension relationship. We conclude that forskolin inhibits smooth muscle contractions by the following two mechanisms: a decrease in Ca²⁺ sensitivity of contractile elements in high K⁺-stimulated muscle and a decrease in [Ca²⁺]_i in histamine-stimulated muscle.     

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.     

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.     

Action potential bursts in central snail neurons elicited by paeonol: roles of ionic currents

Aim:

To investigate the effects of 2′-hydroxy-4′-methoxyacetophenone (paeonol) on the electrophysiological behavior of a central neuron (right parietal 4; RP4) of the giant African snail (Achatina fulica Ferussac).

Methods:

Intracellular recordings and the two-electrode voltage clamp method were used to study the effects of paeonol on the RP4 neuron.

Results:

The RP4 neuron generated spontaneous action potentials. Bath application of paeonol at a concentration of ≥500 μmol/L reversibly elicited action potential bursts in a concentration-dependent manner. Immersing the neurons in Co²⁺-substituted Ca²⁺-free solution did not block paeonol-elicited bursting. Pretreatment with the protein kinase A (PKA) inhibitor KT-5720 or the protein kinase C (PKC) inhibitor Ro 31-8220 did not affect the action potential bursts. Voltage-clamp studies revealed that paeonol at a concentration of 500 μmol/L had no remarkable effects on the total inward currents, whereas paeonol decreased the delayed rectifying K⁺ current (I_KD) and the fast-inactivating K⁺ current (I_A). Application of 4-aminopyridine (4-AP 5 mmol/L), an inhibitor of I_A, or charybdotoxin 250 nmol/L, an inhibitor of the Ca²⁺-activated K⁺ current (I_K(Ca)), failed to elicit action potential bursts, whereas tetraethylammonium chloride (TEA 50 mmol/L), an I_KD blocker, successfully elicited action potential bursts. At a lower concentration of 5 mmol/L, TEA facilitated the induction of action potential bursts elicited by paeonol.

Conclusion:

Paeonol elicited a bursting firing pattern of action potentials in the RP4 neuron and this activity relates closely to the inhibitory effects of paeonol on the I_KD.     

Vasorelaxant and antihypertensive effects of formononetin through endothelium-dependent and -independent mechanisms

Aim:

To investigate the mechanisms underlying the vasorelaxant effect of formononetin, an O-methylated isoflavone, in isolated arteries, and its antihypertensive activity in vivo.

Methods:

Arterial rings of superior mesenteric arteries, renal arteries, cerebral basilar arteries, coronary arteries and abdominal aortas were prepared from SD rats. Isometric tension of the arterial rings was recorded using a myograph system. Arterial pressure was measured using tail-cuff method in spontaneously hypertensive rats.

Results:

Formononetin (1–300 μmol/L) elicited relaxation in arteries of the five regions that were pre-contracted by KCl (60 mmol/L), U46619 (1 μmol/L) or phenylephrine (10 μmol/L). The formononetin-induced relaxation was reduced by removal of endothelium or by pretreatment with L-NAME (100 μmol/L). Under conditions of endothelium denudation, formononetin (10, 30, and 100 μmol/L) inhibited the contraction induced by KCl and that induced by CaCl₂ in Ca²⁺-free depolarized medium. In the absence of extracellular Ca²⁺, formononetin (10, 30, and 100 μmol/L) depressed the constriction caused by phenylephrine (10 μmol/L), but did not inhibit the tonic contraction in response to the addition of CaCl₂ (2 mmol/L). The contraction caused by caffeine (30 mmol/L) was not inhibited by formononetin (100 μmol/L). Formononetin (10 and 100 μmol/L) reduced the change rate of Ca²⁺-fluorescence intensity in response to KCl (50 mmol/L). In spontaneously hypertensive rats, formononetin (5, 10, and 20 mg/kg) slowly lowered the systolic, diastolic and mean arterial pressure.

Conclusion:

Formononetin causes vasodilatation via two pathways: (1) endothelium-independent pathway, probably due to inhibition of voltage-dependent Ca²⁺ channels and intracellular Ca²⁺ release; and (2) endothelium-dependent pathway by releasing NO. Both the pathways may contribute to its antihypertensive effect.     

Electrophysiological mechanisms of sophocarpine as a potential antiarrhythmic agent

Aim:

To examine the electrophysiological effects of sophocarpine on action potentials (AP) and ionic currents of cardiac myocytes and to compare some of these effects with those of amiodarone.

Methods:

Langendorff perfusion set-up was used in isolated guinea pig heart, and responses to sophocarpine were monitored using electrocardiograph. Conventional microelectrode, voltage clamp technique and perforated patch were employed to record fast response AP (fAP), slow response AP (sAP) and ionic currents in guinea pig papillary muscle or rabbit sinus node cells.

Results:

Tachyarrhythmia produced by isoprenaline (15 μmol/L) could be reversed by sophocarpine (300 μmol/L). Sophocarpine (10 μmol/L) decreased the amplitude by 4.0%, maximal depolarization velocity (V_max) of the fAP by 24.4%, and Na⁺ current (I_Na) by 18.0%, while it prolonged the effective refractory period (ERP) by 21.1%. The same concentration of sophocarpine could also decrease the amplitude and V_max of the sAP, by 26.8% and 25.7%, respectively, and attenuated the Ca²⁺ current (I_CaL) and the K⁺ tail current substantially. Comparison of sophocarpine with amiodarone demonstrated that both prolonged the duration and the ERP of fAP and sAP, both decreased the amplitude and V_max of the fAP and sAP, and both slowed the automatic heart rate.

Conclusion:

Sophocarpine could reverse isoprenaline-induced arrhythmia and inhibit I_Na, I_CaL, and I_Kr currents. The electrophysiological effects of sophocarpine are similar to those of amiodarone, which might be regarded as a prospective antiarrhythmic agent.