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.     

Diastolic Ca2+ overload caused by Na+/Ca2+ exchanger during the first minutes of reperfusion results in continued myocardial stunning

The pathogenesis of myocardial stunning caused by brief ischemia and reperfusion remains unclear. The aim of the present study was to investigate the underlying mechanism of myocardial stunning. An isolated cell model of myocardial stunning was firstly established in isolated rat ventricular myocytes exposed to 8 min of simulated ischemia and 30 min of reperfusion, the cardiomyocyte contractile function was used to evaluate myocardial stunning. A diastolic Ca(2+) overload without significant changes in systolic Ca(2+) and the amplitude of Ca(2+) transient during the first 10 min of reperfusion played an important role in the occurrence of myocardial stunning. Decreasing Ca(2+) entry into myocardial cells with low Ca(2+) reperfusion was a very efficient way to prevent myocardial stunning. Diastolic Ca(2+) overload was closely related to the reverse mode of Na(+)/Ca(2+) exchanger (NCX) rather than L-type Ca(2+) channel. The activity of the reverse mode of NCX was found significantly higher at the initial time of reperfusion, and KB-R7943, a selective inhibitor of the reverse mode of NCX, administered at first 10 min of reperfusion rather than at the time of ischemia significantly attenuated myocardial stunning. In addition, NCX inhibition also attenuated the Ca(2+) oscillation and cardiac dysfunction when field stimulus was stopped at first 10 min of reperfusion. These data suggest that one of the important mechanisms of triggering myocardial stunning is diastolic Ca(2+) overload caused by activation of the reverse mode of NCX of cardiomyocytes during the initial period of reperfusion following brief ischemia.     

Effects of SEA0400 and KB-R7943 on Na+/Ca2+ exchange current and L-type Ca2+ current in canine ventricular cardiomyocytes

SEA0400 and KB-R7943 are compounds synthesised to block transsarcolemmal Na⁺/Ca²⁺ exchange current (I_Na/Ca); however, they Have also been shown to inhibit L-type Ca²⁺ current (I_Ca). The potential value of these compounds depends critically on their relative selectivity for I_Na/Ca over I_Ca. In the present work, therefore, the concentration-dependent effects of SEA0400 and KB-R7943 on I_Na/Ca and I_Ca were studied and compared in canine ventricular cardiomyocytes using the whole-cell configuration of the patch clamp technique. SEA0400 and KB-R7943 decreased I_Na/Ca in a concentration-dependent manner, having EC₅₀ values of 111±43 nM and 3.35±0.82 M, when suppressing inward currents, while the respective EC₅₀ values were estimated at 108±18 nM and 4.74±0.69 M in the case of outward current block. SEA0400 and KB-R7943 also blocked I_Ca, having comparable EC₅₀ values (3.6 M and 3.2 M, respectively). At higher concentrations (10 M) both drugs accelerated inactivation of I_Ca, retarded recovery from inactivation and shifted the voltage dependence of inactivation towards more negative voltages. The voltage dependence of activation was slightly modified by SEA0400, but not by KB-R7943. Based on the relatively good selectivity of submicromolar concentrations of SEA0400—but not KB-R7943—for I_Na/Ca over I_Ca, SEA0400 appears to be a suitable tool to study the role of I_Na/Ca in Ca²⁺ handling in canine cardiac cells. At concentrations higher than 1 M, however, I_Ca is progressively suppressed by the compound.     

Diclofenac, a Non-steroidal Anti-inflammatory Drug, Inhibits L-type Ca2+ Channels in Neonatal Rat Ventricular Cardiomyocytes

A non-steroidal anti-inflammatory drug (NSAID) has many adverse effects including cardiovascular (CV) risk. Diclofenac among the nonselective NSAIDs has the highest CV risk such as congestive heart failure, which resulted commonly from the impaired cardiac pumping due to a disrupted excitation-contraction (E-C) coupling. We investigated the effects of diclofenac on the L-type calcium channels which are essential to the E-C coupling at the level of single ventricular myocytes isolated from neonatal rat heart, using the whole-cell voltage-clamp technique. Only diclofenac of three NSAIDs, including naproxen and ibuprofen, significantly reduced inward whole cell currents. At concentrations higher than 3 µM, diclofenac inhibited reversibly the Na⁺ current and did irreversibly the L-type Ca²⁺ channels-mediated inward current (IC₅₀=12.89±0.43 µM) in a dose-dependent manner. However, nifedipine, a well-known L-type channel blocker, effectively inhibited the L-type Ca²⁺ currents but not the Na⁺ current. Our finding may explain that diclofenac causes the CV risk by the inhibition of L-type Ca²⁺ channel, leading to the impairment of E-C coupling in cardiac myocytes.     

Ca2+ paradox injury mediated through TRPC channels in mouse ventricular myocytes

BACKGROUND AND PURPOSE

The Ca²⁺ paradox is an important phenomenon associated with Ca²⁺ overload-mediated cellular injury in myocardium. The present study was undertaken to elucidate molecular and cellular mechanisms for the development of the Ca²⁺ paradox.

EXPERIMENTAL APPROACH

Fluorescence imaging was performed on fluo-3 loaded quiescent mouse ventricular myocytes using confocal laser scanning microscope.

KEY RESULTS

The Ca²⁺ paradox was readily evoked by restoration of the extracellular Ca²⁺ following 10–20 min of nominally Ca²⁺-free superfusion. The Ca²⁺ paradox was significantly reduced by blockers of transient receptor potential canonical (TRPC) channels (2-aminoethoxydiphenyl borate, Gd³⁺, La³⁺) and anti-TRPC1 antibody. The sarcoplasmic reticulum (SR) Ca²⁺ content, assessed by caffeine application, gradually declined during Ca²⁺-free superfusion, which was further accelerated by metabolic inhibition. Block of SR Ca²⁺ leak by tetracaine prevented Ca²⁺ paradox. The Na⁺/Ca²⁺ exchange (NCX) blocker KB-R7943 significantly inhibited Ca²⁺ paradox when applied throughout superfusion period, but had little effect when added for a period of 3 min before and during Ca²⁺ restoration. The SR Ca²⁺ content was better preserved during Ca²⁺ depletion by KB-R7943. Immunocytochemistry confirmed the expression of TRPC1, in addition to TRPC3 and TRPC4, in mouse ventricular myocytes.

CONCLUSIONS AND IMPLICATIONS

These results provide evidence that (i) the Ca²⁺ paradox is primarily mediated by Ca²⁺ entry through TRPC (probably TRPC1) channels that are presumably activated by SR Ca²⁺ depletion; and (ii) reverse mode NCX contributes little to the Ca²⁺ paradox, whereas inhibition of NCX during Ca²⁺ depletion improves SR Ca²⁺ loading, and is associated with reduced incidence of Ca²⁺ paradox in mouse ventricular myocytes.     

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.     

Levosimendan increases L-type Ca(2+) current via phosphodiesterase-3 inhibition in human cardiac myocytes.

To evaluate the potency of levosimendan, a newly developed cardiotonic agent, as a phosphodiesterase-3 inhibitor, we examined its effects on the L-type Ca(2+) current (I(Ca,L)) in single human atrial cells using the whole-cell voltage-clamp method. Levosimendan significantly increased I(Ca,L) in a concentration-dependent manner (E(max), 139.0 +/- 1.8%; EC(50), 54 +/- 3.6 nM). The increase in I(Ca,L) induced by 1 microM levosimendan was significantly greater in human atrial cells (136.7 +/- 11.0%, n=8) than in rabbit atrial cells (23.5 +/- 3.5%, n=6) (depolarization to +10 mV in each case). In rat atrial and ventricular cells, I(Ca,L) was unaffected by 1-10 microM levosimendan. These results indicate that the selective phosphodiesterase-3 inhibitor levosimendan increases cardiac-cell I(Ca,L) significantly more strongly in human than in rabbit and rat. It seems likely that the positive inotropic effect of levosimendan on the human myocardium depends on an increase in I(Ca,L) that is modulated by adenosine 3'5'-cyclic monophosphate (cAMP)-dependent phosphorylation.     

Nonylphenol,an environmental estrogen,affects voltage-gated K currents and L-type Ca currents in a non-monotonic manner in GH3 pituitary cells

We have investigated the characteristics of voltage-gated K⁺ channels and L-type Ca²⁺ channels in GH₃ rat pituitary cells and the effects of the xenoestrogen (XEs) nonylphenol (NP) on these ion channel currents. Our results have shown that the lower concentrations (10⁻¹⁵–10⁻¹⁴ M) of NP decreased the amplitudes of voltage-gated K⁺ currents (I_Kv) and activated L-type Ca²⁺ currents (I_Ca-L) by reducing half-activation membrane potentials of activation kinetics curves. However, the higher concentrations (10⁻¹⁰–10⁻⁹ M) of NP increased the amplitudes of I_Kv and inhibited I_Ca-L by reducing the peak values of I_Ca-L. Thus, NP affects I_Kv and I_Ca-L in an opposite and non-monotonic manner.     

Phenytoin partially antagonized L-type Ca2+ current in glucagon-secreting tumor cells (ITC-1)

Summary Transmembrane Ca²⁺ currents were investigated by means of a whole-cell clamp technique in a hamster glucagon-secreting tumor cell line (ITC-1). Two types of Ca²⁺ current were identified in ITC-1 cells. The low-threshold and transient (T-type) current became detectable above the potential level around –60 mV and decayed rapidly with an inactivation time constant of 95 ms (at –40 mV and 23°C), while the high-threshold and long-lasting (L-type) one was activated by depolarization more positive to –30 mV with non-inactivating kinetics. The voltage dependence and kinetics of these currents were identical to those reported in guinea-pig pancreatic ₂ cells. Both currents were augmented by equimolar substitution of Ca²⁺ with Ba²⁺ and completely abolished by adding 1 M La³⁺. Phenytoin, a well known anti-epileptic drug and a postulated T-type specific Ca²⁺ current antagonist, surprisingly blocked the L-type current without affecting the T-type current in ITC-1 cells. While phenytoin antagonized the L-type Ba²⁺ current selectively, 60% of the current remained even in supramaximal concentration range over 500 M. The residual component of the L-type current was completely abolished by adding nifedipine.     

Protection of ischemic rat spinal cord white matter: Dual action of KB-R7943 on Na+/Ca2+ exchange and L-type Ca2+ channels

The effect of the Na+/Ca(2+)-exchange inhibitor KB-R7943 was investigated in spinal cord dorsal column ischemia in vitro. Oxygen/glucose deprivation at 37 degrees C for 1 h causes severe injury even in the absence of external Ca2+. KB-R7943 was very protective in the presence and absence of external Ca2+ implicating mechanisms in addition to extracellular Ca2+ influx through Na+/Ca(2+)-exchange, such as activation of ryanodine receptors by L-type Ca2+ channels. Indeed, blockade of L-type Ca2+ by nimodipine confers a certain degree of protection of dorsal column against ischemia; combined application of nimodipine and KB-R7943 was not additive suggesting that KB-R7943 may also act on Ca2+ channels. KB-R7943 reduced inward Ba2+ current with IC50 = 7 microM in tsA-201 cells expressing Ca(v)1.2. Moreover, nifedipine and KB-R7943 both reduced depolarization-induced [Ca2+]i increases in forebrain neurons and effects were not additive. Nimodipine or KB-R7943 also reduced ischemic axoplasmic Ca2+ increase, which persisted in 0Ca2+/EGTA perfusate in dorsal column during ischemia. While KB-R7943 cannot be considered to be a specific Na+/Ca2+ exchange inhibitor, its profile makes it a very useful neuroprotectant in dorsal columns by: reducing Ca2+ import through reverse Na+/Ca2+ exchange; reducing influx through L-type Ca2+ channels, and indirectly inhibiting Ca2+ release from the ER through activation of ryanodine receptors.     

Mechanisms of Na+ and Ca2+ influx into respiratory neurons during hypoxia

Changes in intracellular Na+ and Ca2+ in inspiratory neurons of neonatal mice were examined by using ion-selective fluorescent indicator dyes SBFI and fura-2, respectively. Both [Na+]i and [Ca2+]i signals showed rhythmic elevations, correlating with the inspiratory motor output. Brief (2-3 min) hypoxia, induced initial potentiation of rhythmic transients followed by their depression. During hypoxia, the basal [Na+]i and [Ca2+]i levels slowly increased, reflecting development of an inward current (Im). By antagonizing specific mechanisms of Na+ and Ca2+ transport we found that increases in [Na+]i, [Ca2+]i and Im due to hypoxia are suppressed by CNQX, nifedipine, riluzole and flufenamic acid, indicating contribution of AMPA/kainate receptors, persistent Na+ channels, L-type Ca2+ channels and Ca2+-sensitive non-selective cationic channels, respectively. The blockers decreased also the amplitude of the inspiratory bursts. Modification of mitochondrial properties with FCCP and cyclosporine A decreased [Ca2+]i elevations due to hypoxia by about 25%. After depletion of internal Ca2+ stores with thapsigargin, the blockade of NMDA receptors, Na+/K+ pump, Na+/H+ and Na+/Ca2+ exchange, the hypoxic response was not changed. We conclude that slow [Na+]i and [Ca2+]i increases in inspiratory neurons during hypoxia are caused by Na+ and Ca2+ entry due to combined activation of persistent Na+ and L-type Ca2+ channels and AMPA/kainate receptors.     

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.     

Induces vasodilatation of rat mesenteric arteryin vitro mainly by inhibiting receptor-mediated Ca2+-influx and Ca2+-release

The purpose of this study was to investigate the effect of atropine on peripheral vasodilation and the mechanisms involved. The isometric tension of rat mesenteric artery rings was recorded in vitro on a myograph. The results showed that atropine, at concentrations greater than 1 microM, relaxed the noradrenalin (NA)-precontracted rat mesenteric artery in a concentration-dependent manner. Atropine-induced vasodilatation was mediated, in part, by an endothelium-dependent mechanism, to which endothelium-derived hyperpolarizing factor may contribute. Atropine was able to shift the NA-induced concentration-response curve to the right, in a non-parallel manner, suggesting the mechanism of atropine was not mediated via the (alpha1-adrenoreceptor. The beta-adrenoreceptor and ATP sensitive potassium channel, a voltage dependent calcium channel, were not involved in the vasodilatation. However, atropine inhibited the contraction derived from NA and CaCI2 in Ca(2+)-free medium, in a concentration dependent manner, indicating the vasodilatation was related to the inhibition of extracellular Ca2+ influx through the receptor-operated calcium channels and intracellular Ca2+ release from the Ca2+ store. Atropine had no effect on the caffeine-induced contraction in the artery segments, indicating the inhibition of intracellular Ca2+ release as a result of atropine most likely occurs via the IP3 pathway rather than the ryanodine receptors. Our results suggest that atropine-induced vasodilatation is mainly from artery smooth muscle cells due to inhibition of the receptor-mediated Ca(2+)-influx and Ca(2+)-release, and partly from the endothelium mediated by EDHF.     

PKC-Independent Stimulation of Cardiac Na+/Ca2+ Exchanger by Staurosporine

[Ca²⁺]_i transients by reverse mode of cardiac Na⁺/Ca²⁺ exchanger (NCX1) were recorded in fura-2 loaded BHK cells with stable expression of NCX1. Repeated stimulation of reverse NCX1 produced a long-lasting decrease of Ca²⁺ transients (''rundown''). Rundown of NCX1 was independent of membrane PIP₂ depletion. Although the activation of protein kinase C (PKC) was observed during the Ca²⁺ transients, neither a selective PKC inhibitor (calphostin C) nor a PKC activator (PMA) changed the degrees of rundown. By comparison, a non-specific PKC inhibitor, staurosporine (STS), reversed rundown in a dose-dependent and reversible manner. The action of STS was unaffected by pretreatment of the cells with calphostin C, PMA, or forskolin. Taken together, the results suggest that the stimulation of reverse NCX1 by STS is independent of PKC and/or PKA inhibition.     

Functional, morphological and molecular characterization of bladder dysfunction in streptozotocin-induced diabetic mice: evidence of a role for L-type voltage-operated Ca2+ channels

BACKGROUND AND PURPOSE

Diabetic cystopathy is one of the most common and incapacitating complications of diabetes mellitus. This study aimed to evaluate the functional, structural and molecular alterations of detrusor smooth muscle (DSM) in streptozotocin-induced diabetic mice, focusing on the contribution of Ca²⁺ influx through L-type voltage-operated Ca²⁺ channels (L-VOCC).

EXPERIMENTAL APPROACH

Male C57BL/6 mice were injected with streptozotocin (125 mg·kg⁻¹). Four weeks later, contractile responses to carbachol, α,β-methylene ATP, KCl, extracellular Ca²⁺ and electrical-field stimulation were measured in urothelium-intact DSM strips. Cystometry and histomorphometry were performed, and mRNA expression for muscarinic M₂/M₃ receptors, purine P2X1 receptors and L-VOCC in the bladder was determined.

KEY RESULTS

Diabetic mice exhibited higher bladder capacity, frequency, non-void contractions and post-void pressure. Increased bladder weight, wall thickness, bladder volume and neural tissue were observed in diabetic bladders. Carbachol, α,β-methylene ATP, KCl, extracellular Ca²⁺ and electrical-field stimulation all produced greater DSM contractions in diabetic mice. The L-VOCC blocker nifedipine almost completely reversed the enhanced DSM contractions in bladders from diabetic animals. The Rho-kinase inhibitor Y27632 had no effect on the enhanced carbachol contractions in the diabetic group. Expression of mRNA for muscarinic M₃ receptors and L-VOCC were greater in the bladders of diabetic mice, whereas levels of M₂ and P2X1 receptors remained unchanged.

CONCLUSIONS AND IMPLICATIONS

Diabetic mice exhibit features of urinary bladder dysfunction, as characterized by overactive DSM and decreased voiding efficiency. Functional and molecular data suggest that overactive DSM in diabetes is the result of enhanced extracellular Ca²⁺ influx through L-VOCC.     

高胆固醇血症致大鼠心室肌细胞L-型钙电流失衡的机制

目的研究高胆固醇血症(HC)对大鼠心室肌细胞L-型钙电流(ICa-L)以及细胞内钙浓度[Ca2+]i的影响。方法Wistar大鼠随机分为高胆固醇血症组和对照组各6只,分别给予高胆固醇饲料和普通饲料饲养4wk后,检测血脂;酶解法急性分离大鼠单个心肌细胞,采用全细胞膜片钳技术和激光扫描共聚焦显微镜观察高胆固醇血症对大鼠心室肌细胞ICa-L以及[Ca2+]i的变化。结果喂高胆固醇饲料4wk后,血清总胆固醇较正常对照组明显增高(P<0·01),甘油三脂无明显变化。膜片钳显示:高胆固醇血症大鼠各实验电压L-型钙电流电流密度均减少。在实验电压为0mV时,大鼠心室肌细胞L-型钙电流密度从正常对照组(-8·56±1·29)pA/pF减少到高胆固醇血症组(-5·24±0·90)pA/pF。激光共聚焦显示:在静息状态下,[Ca2+]i升高,峰值荧光强度由211·88±9·08增加至458·63±23·50(P<0·01)。结论高胆固醇血症可导致细胞内钙超载,使L-型钙通道电流失衡,可能是诱导心律失常的原因之一。     

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.     

广枣总黄酮对大鼠心室肌细胞IC_a、I_(to)和细胞[Ca~(2+)]_i的影响

目的　观察广枣总黄酮对大鼠心室肌细胞L 型钙通道电流 (ICa)和瞬时外向钾通道电流 (Ito)以及对心肌细胞内游离钙浓度 ([Ca2 + ]i)的影响 ,探讨其抗心律失常作用机制。方法　全细胞膜片钳记录大鼠心室肌细胞ICa、Ito,激光共聚焦显微镜观察细胞 [Ca2 + ]i 的变化。结果　在钳制电压- 4 0mV ,实验电压 - 4 0～ +5 0mV时 ,广枣总黄酮 10 0mg·L-1对心室肌细胞ICa无显著影响 ;在钳制电压 - 6 0mV ,实验电压 - 4 0～ +5 0mV时显著抑制瞬时外向钾通道Ito(P <0 0 5 ) ;而激光共聚焦显微镜结果显示广枣总黄酮在 5 0、10 0、2 0 0mg·L-1却降低缺氧复氧心肌细胞收缩期和静息期[Ca2 + ]i 的浓度。结论　广枣总黄酮对心肌细胞ICa无显著影响 ,可显著抑制瞬时外向钾通道Ito,并可明显降低心肌细胞收缩期和静息期细胞 [Ca2 + ]i 浓度。这可能是其抗心律失常和保护缺血心肌的主要作用机制。     

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.     

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.