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.     

Relaxant Effect of Spermidine on Acethylcholine and High K+-induced Gastric Contractions of Guinea-Pig

In our previous study, we found that spermine and putrescine inhibited spontaneous and acetylcholine (ACh)-induced contractions of guinea-pig stomach via inhibition of L-type voltage-dependent calcium current (VDCC_L). In this study, we also studied the effect of spermidine on mechanical contractions and calcium channel current (I_Ba), and then compared its effects to those by spermine and putrescine. Spermidine inhibited spontaneous contraction of the gastric smooth muscle in a concentration-dependent manner (IC₅₀=1.1±0.11 mM). Relationship between inhibition of contraction and calcium current by spermidine was studied using 50 mM high K⁺-induced contraction: Spermidine (5 mM) significantly reduced high K⁺ (50 mM)-induced contraction to 37±4.7% of the control (p<0.05), and inhibitory effect of spermidine on I_Ba was also observed at a wide range of test potential in current/voltage (I/V) relationship. Pre- and post-application of spermidine (5 mM) also significantly inhibited carbachol (CCh) and ACh-induced initial and phasic contractions. Finally, caffeine (10 mM)-induced contraction which is activated by Ca²⁺-induced Ca²⁺ release (CICR),'' was also inhibited by pretreatment of spermidine (5 mM). These findings suggest that spermidine inhibits spontaneous and CCh-induced contraction via inhibition of VDCC_L and Ca²⁺ releasing mechanism in guinea-pig stomach.     

四肽FMRFa对大鼠心室肌Na+/Ca2+交换的抑制

目的　研究四肽FMRFa对大鼠单个心室肌细胞Na⁺/Ca²⁺交换的作用。方法　用膜片钳全细胞记录法测定成年大鼠心室肌细胞Na⁺/Ca²⁺交换电流(I_Na⁺/Ca²⁺)和其他离子通道电流。结果　FMRFa对大鼠心室肌细胞I_Na⁺/Ca²⁺呈浓度依赖性抑制,100μmol·L^-1浓度时抑制内向和外向I_Na⁺/Ca²⁺密度分别达60.1%和56.5%,对内向电流及外向电流的IC₅₀分别为20μmol·L^-1和34μmol·L^-1。FMRFa5μmol·L^-1抑制I_Na⁺/Ca²⁺内向和外向电流密度分别为38.7%和34.9%,但FMRFa5μmol·L^-1及20μmol·L^-1对L型钙电流、钠电流、瞬时外向电流和内向整流钾电流均无显著抑制作用。结论　FMRFa对大鼠心室肌细胞是一个特异性Na⁺/Ca²⁺交换抑制剂。     

In vitro vasodilator mechanisms of the indole alkaloids rhynchophylline and isorhynchophylline,isolated from the hook of<Emphasis Type="Italic"> Uncaria rhynchophylla</Emphasis> (Miquel)

Rhynchophylline (Rhy) and isorhynchophylline (Isorhy), indole alkaloids from Uncaria hooks, reportedly exert hypotensive and vasodilatory effects, but the mechanism of action is unclear. We therefore investigated the relaxant effects of these two isomeric alkaloids in rat arteries in vitro, in particular in respect of the various functional Ca²⁺ pathways. Both Rhy and Isorhy relaxed aortic rings precontracted with phenylephrine (PE, 1 µM) in a dose-dependent manner (3–300 µM). Removal of endothelium and preincubation with L-NAME (300 µM) slightly inhibited but did not prevent the relaxant response. These results indicate that Rhy and Isorhy act largely in an endothelium-independent manner. Unlike nicardipine, both alkaloids not only inhibited the contraction induced by 60 mM KCl (IC₅₀ 20–30 µM), but also that induced by PE and U46619, albeit to a lesser extent (IC₅₀ 100 and 200 µM, respectively). These results suggest that Rhy and Isorhy may act via multiple Ca²⁺ pathways. In contrast to their inhibitory effects on KCl-induced and receptor-mediated contractions, where both isomers were comparably potent, Rhy was more potent than Isorhy at higher concentrations (>100 µM) in inhibiting both caffeine (25 mM)- and cyclopiazonic acid (CPA, 30 µM)-induced contractions. Similar results observed with caffeine in Ca²⁺-containing medium were also observed in Ca²⁺-free medium. However, 0.1–0.3 µM nicardipine (which completely inhibited KCl-induced contraction) had no significant inhibitory effect on CPA-induced contractions. Taken together, these results indicate discrimination between these two isomers with respect to Ca²⁺-induced Ca²⁺ release and non-L-type Ca²⁺ channel, but not for IP₃-induced Ca²⁺ release and L-type Ca²⁺ channels. Similar relaxant responses to KCl- and caffeine-induced contractions were seen when these two alkaloids were tested on the smaller mesenteric and renal arteries. In conclusion, the vasodilatory effects of Rhy and Isorhy are largely endothelium independent and are mediated by L-type Ca²⁺ channels. At higher concentrations, they also affect other Ca²⁺-handling pathways, although to a lesser extent. While there is no discrimination between the two isomers with respect to the contraction induced by KCl or agonists (PE and U46619), differential effects between Rhy and Isorhy were seen on caffeine- and CPA-induced contractions.     

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.     

K+ channel openers,cromakalim and Ki4032, inhibit agonist-induced Ca2+ release in canine coronary artery

Summary The effects of K⁺ channel openers, cromakalim and an acetoxyl derivative of KRN 2391 (Ki 4032), were studied on force of contraction, increases in intracellular calcium concentration ([Ca²⁺]_i) measured by fura-2 and inositol 1,4,5-trisphosphate (IP₃) production induced by the thromboxane A₂ analogue, U46619, in canine coronary arteries. Upon single dose applications of U46619 at 300 nmol/l, phasic and tonic increases in [Ca²⁺]_i and force were seen, which were almost abolished by cromakalim (10 mol/l) and Ki4032 (100 mol/l).In the absence of extracellular Ca²⁺, U46619 induced a transient increase in [Ca²⁺]_i with a contraction. Cromakalim (0.01–10 mol/l) and Ki4032 (0.1–100 mol/l) concentration-dependently inhibited the increases in [Ca²⁺]_i and contraction. The inhibitory effects of cromakalim and Ki4032 were blocked by the K⁺ channel blocker tetrabutylammonium (TBA) and counteracted by 20 mmol/l KCl-induced depolarization. Cromakalim and Ki4032 did not affect caffeine-induced Ca²⁺ release. Cromakalim reduced U46619-induced IP₃ production significantly and TBA blocked this inhibitory effect. These results suggest that the hyperpolarization of the plasma membrane by K⁺ channel openers inhibits the production of IP₃ and Ca ²⁺ release from intracellular stores related to stimulation of the thromboxane A₂ receptor.Correspondence to T. Yanagisawa at the above address     

Endothelin-1 induced contraction of rat aorta: contributions made by Ca2+ influx and activation of contractile apparatus associated with no change in cytoplasmic Ca2+ level

Summary The modes by which Endothelin-1 (ET) induces Ca²⁺-influx and the relative functional importance of the different sources of Ca²⁺ for ET-induced contraction were studied using fura 2-loaded and unloaded rat aortic strips. ET caused an increase in the cytosolic free Ca²⁺ level ([Ca²⁺]_i) followed by a tonic contraction in Ca²⁺-containing solution, and produced a transient elevation of [Ca²⁺]_i followed by a small sustained contraction in Ca²⁺-free medium. ET also stimulated ⁴⁵Ca influx into La²⁺-inaccessible fraction significantly. With the same change of [Ca²⁺]_i, ET caused a larger tension than that induced by high K. ET-induced contraction and [Ca²⁺]_i elevation were not significantly inhibited by 0.1–0.3 M nicardipine which nearly abolished the contraction and [Ca⁺]_i elevation produced by high K. During treatment of the strips with high K, addition of ET induced further increases in [Ca²⁺]_i and muscle tension, and vice versa. In Ca²⁺-free medium, ET-induced contraction was influenced neither by ryanodine-treatment nor by high K-treatment, although the former attenuated and the latter potentiated the [Ca²⁺]_i transient induced by ET. Further, the ET-induced sustained contraction under Ca²⁺-free conditions began to develop after the [Ca²⁺]_i level returned to the baseline. Thus, it seems that the Ca²⁺ released from the ryanodine-sensitive and -insensitive Ca²⁺ stores by ET may provide only a minor or indirect contribution, if any, to the tension development. ET might cause a contraction mainly by stimulating Ca²⁺-influx through Ca²⁺ channel(s) other than voltage-dependent Ca²⁺ channels in character, and by increasing the sensitivity of the contractile filaments to Ca²⁺ or activating them Ca²⁺-independently.Visiting from Zun Yi Medical College, China Send offprint requests to I. Takayanagi at the above address     

Inhibitory effects of sevoflurane on pacemaking activity of sinoatrial node cells in guinea-pig heart

BACKGROUND AND PURPOSE

The volatile anaesthetic sevoflurane affects heart rate in clinical settings. The present study investigated the effect of sevoflurane on sinoatrial (SA) node automaticity and its underlying ionic mechanisms.

EXPERIMENTAL APPROACH

Spontaneous action potentials and four ionic currents fundamental for pacemaking, namely, the hyperpolarization-activated cation current (I_f), T-type and L-type Ca²⁺ currents (I_Ca,T and I_Ca,L, respectively), and slowly activating delayed rectifier K⁺ current (I_Ks), were recorded in isolated guinea-pig SA node cells using perforated and conventional whole-cell patch-clamp techniques. Heart rate in guinea-pigs was recorded ex vivo in Langendorff mode and in vivo during sevoflurane inhalation.

KEY RESULTS

In isolated SA node cells, sevoflurane (0.12–0.71 mM) reduced the firing rate of spontaneous action potentials and its electrical basis, diastolic depolarization rate, in a qualitatively similar concentration-dependent manner. Sevoflurane (0.44 mM) reduced spontaneous firing rate by approximately 25% and decreased I_f, I_Ca,T, I_Ca,L and I_Ks by 14.4, 31.3, 30.3 and 37.1%, respectively, without significantly affecting voltage dependence of current activation. The negative chronotropic effect of sevoflurane was partly reproduced by a computer simulation of SA node cell electrophysiology. Sevoflurane reduced heart rate in Langendorff-perfused hearts, but not in vivo during sevoflurane inhalation in guinea-pigs.

CONCLUSIONS AND IMPLICATIONS

Sevoflurane at clinically relevant concentrations slowed diastolic depolarization and thereby reduced pacemaking activity in SA node cells, at least partly due to its inhibitory effect on I_f, I_Ca,T and I_Ca,L. These findings provide an important electrophysiological basis of alterations in heart rate during sevoflurane anaesthesia in clinical settings.     

Noradrenergic vasoconstriction of pig prostatic small arteries

The current study investigated the distribution of adrenergic nerves and the action induced by noradrenaline (NA) in pig prostatic small arteries. Noradrenergic innervation was visualized using an antibody against dopamine-beta-hydroxylase (DBH), and the NA effect was studied in small arterial rings mounted in microvascular myographs for isometric force recordings. DBH-immunoreactive nerve fibers were located at the adventitia and the adventitia-media border of the vascular wall. Electrical field stimulation (EFS, 1-32 Hz) evoked frequency-dependent contractions that were reduced by guanethidine and prazosin (adrenergic neurotransmission and α₁-adrenoceptors blockers, respectively) and by the α₂-adrenoceptor agonist UK 14,304. The α₂-adrenoceptor antagonist rauwolscine reversed the UK 14,304-produced inhibition. NA produced endothelium-independent contractions that were antagonized with low estimated affinities and Schild slopes different from unity by prazosin and the α_1A-adrenoceptor antagonist N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-α-α-dimethyl-1H-indole-3-ethanamine (RS 17053). The α_1A-adrenoceptor antagonist 5-methyl-3-[3-[4-[2-(2,2,2,-trifluoroethoxy) phenyl]-1-piperazinyl]propyl]-2,4-(1H)-pyrimidinedione (RS 100329), which also displays high affinity for α_1L-adrenoceptors, and the α_1L-adrenoceptor antagonist tamsulosin, which also has high affinity for α_1A- and α_1D-adrenoceptors, induced rightward shifts with high affinity of the contraction-response curve to NA. The α_1D-adrenoceptor antagonist 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-ethyl]8-azaspiro[4,5]decane-7,9-dione dihydrochloride (BMY 7378) failed to modify the NA contractions that were inhibited by extracellular Ca²⁺ removal and by voltage-activated (L-type) Ca²⁺ channel blockade. These data suggest that pig prostatic resistance arteries have a rich noradrenergic innervation; and NA, whose release is modulated by prejunctional α₂-adrenoceptors, evokes contraction mainly through activation of muscle α_1L-adrenoceptors coupled to extracellular Ca²⁺ entry via voltage (L-type)- and non-voltage-activated Ca²⁺ channels.     

Effects of dopamine on L-type Ca2+ current in single atrial and ventricular myocytes of the rat

1. The effects of dopamine on the L-type Ca²⁺ current (I_Ca,L) of both atrial and ventricular single myocytes and on the force of contraction of atrial trabeculae in rat heart were investigated.
2. Dopamine increased atrial I_Ca,L at concentrations higher than 1 μM, but had little or no effect on I_Ca,L at lower concentrations. The increase in I_Ca,L at high concentrations was reversed by propranolol and acetylcholine, but not by phentolamine. Activation and inactivation kinetics of I_Ca,L were not altered by dopamine.
3. In rat ventricular myocytes in which the D₄ receptor mRNA does not express, dopamine (20–100 μM) also increased the I_Ca,L amplitude and propranolol reversed this effect.
4. Clozapine, a potent D₄ receptor antagonist, blocked the augmenting effect of dopamine on I_Ca,L. However, this effect could be explained by β-antagonism, since clozapine also inhibited the isoprenaline effect.
5. In the atrial trabeculae, the increase in contraction by dopamine (1 to 30 μM) was reversed by 1 μM propranolol, but not by 2 μM phentolamine. Low doses of dopamine (0.01 to 0.3 μM) did not affect the contraction in the controls or during a modest stimulation of the β-adrenoceptor with 0.01 μM isoprenaline.
6. These results indicate that the positive inotropic action of dopamine is mediated through direct stimulation of the β-adrenoceptor in both atrial and ventricular myocytes. Involvement of D₄ receptor appears unlikely in the regulation of the atrial contraction.

     

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.     

Antagonistic actions of S(−)-Bay K 8644 on cyclic nucleotide-induced inhibition of voltage-dependent Ba2+ currents in guinea pig gastric antrum

(±)-Bay K 8644, a conventional racemic mixture of Bay K 8644, is widely used as an L-type Ca²⁺ channel agonist. Although interactions between Bay K 8644 and cyclic nucleotide have been described, they have not been properly characterized. We have investigated whether two optical isomers of Bay K 8644 (i.e., R(+)- and S(−)-Bay K 8644) modify cyclic nucleotide (cAMP and cGMP)-induced inhibitory effects on nifedipine-sensitive voltage-dependent Ba²⁺ currents (I _Ba) recorded from guinea pig gastric myocytes. Conventional whole-cell recordings were used to compare the effects of R(+)-Bay K 8644 and S(−)-Bay K 8644 on I _Ba. S(−)-Bay K 8644 enhanced the peak amplitude of I _Ba evoked by depolarizing pulses to +10 mV from a holding potential of −70 mV in a concentration-dependent manner (EC₅₀ = 32 nM), while R(+)-Bay K 8644 inhibited I _Ba (IC₅₀ = 975 nM). When R(+)-Bay K 8644 (0.5 μM) was applied, I _Ba was suppressed to 71 ± 10% of control. In the presence of R(+)-Bay K 8644 (0.5 μM), additional application of forskolin and sodium nitroprusside (SNP) further inhibited I _Ba. Conversely, in the presence of S(−)-Bay K 8644 (0.5 μM), subsequent application of forskolin and SNP did not affect I _Ba. Similarly, in the presence of 0.5 μM S(−)-Bay K 8644, db-cAMP and 8-Br-cGMP had no effect on I _Ba. These results indicate that S(−)-Bay K 8644, but not R(+)-Bay K 8644, can prevent the inhibitory actions of two distinct cyclic nucleotide pathways on I _Ba in gastric myocytes of the guinea pig antrum.     

Mechanisms underlying the slow onset of action of a new dihydropyridine,NZ-105, on a cultured smooth muscle cell line

Summary The inhibitory effect of a new dihydropyridine derivative, (±)-2-[benzyl(phenyl)amino]ethyl-1,4-dihydro-2,6-dimethyl-5-(5,5-dimethyl-2-oxo-1,3,2-dioxaphosphorinan-2-yl)-4-(3-nitrophenyl)-3-pyridinecarboxylate hydrochloride (NZ-105), on whole cell Ca²⁺ current (I_Ca) in cultured vascular smooth muscle cells was investigated with the patch clamp technique. NZ-105 blocked I_Ca in a concentration-dependent manner when the command pulse ranged from +10 mV to –50 mV. The inhibitory effect of NZ-105 appeared at concentrations higher than 10 mol/l and it blocked I_Ca completely at a concentration of 1 nmol/l. The concentration which produced the half-maximal inhibitory effect was estimated to be around 20 mol/l. NZ-105 (500 pmol/l) completely blocked I_Ca elicited by depolarization to + 10 mV at a holding potential of –40 mV, whereas it blocked I_Ca by only 67% at a holding potential of –90 mV. NZ-105 (100 mol/l) shifted the steady-state inactivation curve by 40 mV to more negative potentials without affecting its slope factor. The blocking time constant of 500 mol/l NZ-105 was 57.6 + 9.9 s at a holding potential of –70 mV. These results indicate that NZ-105 has characteristics typical of dihydropyridines and binds to Ca²⁺ channels of vascular smooth muscle cells with a high affinity. They also suggested that the slow onset of its action is due to the slow binding of the drug to Ca²⁺ channels. Send offprint requests to S. Kokubun at the above address     

Accelerated inactivation of cardiac L-type calcium channels triggered by anaesthetic-induced preconditioning

Background and purpose:

Cardioprotection against ischaemia by anaesthetic-induced preconditioning (APC) is well established. However, the mechanism underlying Ca²⁺ overload attenuation by APC is unknown. The effects of APC by isoflurane on the cardiac L-type Ca channel were investigated.

Experimental approach:

In a model of in vivo APC, Wistar rats were exposed to isoflurane (1.4%), delivered via a vaporizer in an enclosure, prior to thoracotomy. The Dahl S rats were similarly preconditioned to determine strain-dependent effects. Whole-cell patch clamp using cardiac ventricular myocytes was used to determine the L-type Ca²⁺ current (I_Ca,L) characteristics and calmodulin (CaM) levels were determined by Western blot analysis. Cytosolic Ca²⁺ levels were monitored using fluo-4-AM. Action potential (AP) simulations examined the effects of APC.

Key results:

In Wistar rats, APC significantly accelerated I_Ca,L inactivation kinetics. This was abolished when external Ca²⁺ was replaced with Ba²⁺, suggesting that Ca²⁺-dependent inactivation of I_Ca,L was modulated by APC. Expression levels of CaM, a determinant of I_Ca,L inactivation, were not affected. Attenuation of cytosolic Ca²⁺ accumulation following oxidative stress was observed in the APC group. Simulations showed that the accelerated inactivation of I_Ca,L resulted in a shortening of the AP duration. The Dahl S rat strain was resistant to APC and changes in I_Ca,L inactivation were not observed in cardiomyocytes prepared from these rats.

Conclusions and implications:

APC triggered persistent changes in the inactivation of cardiac L-type Ca channels. This can potentially lead to a reduction in Ca²⁺ influx and attenuation of Ca²⁺ overload during ischaemia/reperfusion.     

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.     

A novel secretagogue increases cardiac contractility by enhancement of L-type Ca current

N′1-(3,3,6,8-tetramethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yliden)-2-cyanoethanohydrazide (TTYC) increases secretion of glucagon-like peptide-1 and intracellular Ca²⁺ concentration in GLUTag cells. The purpose of the present study was to examine if TTYC exerts positive inotropic effects on isolated rabbit ventricular myocytes and in vivo heart in anesthetized rats, and if so to further define the potential mechanism of action. Contractility was assessed in vitro using changes in fractional shortening (FS) of myocyte sarcomere length and in vivo using changes in the velocity of left ventricular pressure. Changes in L-type Ca²⁺ current of ventricular myocytes were evaluated using whole-cell voltage-clamp techniques. TTYC increased FS of myocyte sarcomere length in a concentration-dependent manner. The positive inotropic effect was not abrogated by β-adrenergic blockade (propranolol) or protein kinase A inhibition. TTYC enhanced peak L-type Ca²⁺ current in a voltage-dependent manner (current amplitudes increased by 4.0-fold at -10 mV and 1.5-fold at +10 mV). Voltage-dependence of steady-state activation of L-type Ca²⁺ current was shifted by 15 mV in the negative direction. Inactivation time course of the L-type Ca²⁺ currents at voltages of −10 to 20 mV was significantly slowed by 0.3 μM TTYC. In vivo studies demonstrated that TTYC increased cardiac contractility in a dose-dependent manner. In conclusion, TTYC is a novel L-type Ca²⁺ current activator with positive cardiac inotropic effects. Negative shifting of the voltage-dependence of L-type Ca²⁺ current activation and reduced inactivation are two mechanisms responsible for the enhanced L-type Ca²⁺ current that contribute to the positive inotropic effects.     

Pharmacological evidence for altered src kinase regulation of <Emphasis Type="Italic">I</Emphasis><Subscript>Ca,L</Subscript> in patients with chronic atrial fibrillation

A reduction in l-type Ca²⁺ current (I _Ca,L) contributes to electrical remodeling in chronic atrial fibrillation (AF). Whether the decrease in I _Ca,L is solely due to a reduction in channel proteins remains controversial. Protein tyrosine kinases (PTK) have been described as potent modulators of I _Ca,L in cardiomyocytes. We studied α_1C l-type Ca²⁺ channel subunit expression and the regulation of I _Ca,L by PTK in chronic AF using PTK inhibitors: genistein, a nonselective inhibitor of PTK, and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-3,4-d-pyrimidine (PP1), a selective inhibitor of src kinases. Furthermore, type-1 and type-2A protein phosphatase activity was measured with phosphorylase as substrate in whole-cell lysates derived from atrial tissue of AF patients. Right atrial appendages were obtained from patients undergoing open-heart surgery. Protein levels of α_1C l-type Ca²⁺ channel subunit were determined using Western blot analysis and normalized to the protein amounts of calsequestrin as internal control. The protein concentrations of α_1C did not differ between AF and sinus rhythm (SR; α_1C/calsequestrin: 1.0 ± 0.1 and 1.2 ± 0.2, respectively, n = 8 patients). In cardiomyocytes from patients in SR (n = 20 patients), genistein and PP1 both evoked similar increases in I _Ca,L from 3.0 ± 0.3 to 6.1 ± 0.8 pA/pF and from 2.8 ± 0.4 to 6.1 ± 0.6 pA/pF, respectively. In cells from AF patients (n = 10 patients), basal I _Ca,L was significantly lower. In this case, genistein lead to the same relative increase in I _Ca,L as in SR cells (from 1.46 ± 0.30 to 3.2 ± 1.0 pA/pF), whereas no increase was elicited by PP1 suggesting impaired regulation of I _Ca,L by src kinases in AF. Total and type 1 and type 2A-related phosphatase activities were higher in tissue from patients with chronic AF compared to SR (4.8 ± 0.4, 2.1 ± 0.2, and 2.7 ± 0.4 nmol/mg/min and 3.6 ± 0.4, 1.3 ± 0.2, and 2.4 ± 0.3 nmol/mg/min, respectively, n = 7 patients per group). Downregulation of I _Ca,L in AF is not due to a reduction in l-type Ca²⁺ channel protein expression. Indirect evidence for an impaired src kinase regulation of I _Ca,L together with an increased phosphatase activity suggests that a complex alteration in the kinase/phosphatase balance leads to I _Ca,L dysregulation in chronic AF.     

Lysophosphatidylcholine Increases Ca2+ Current via Activation of Protein Kinase C in Rabbit Portal Vein Smooth Muscle Cells

Lysophosphatidylcholine (LPC), a metabolite of membrane phospholipids by phospholipase A₂, has been considered responsible for the development of abnormal vascular reactivity during atherosclerosis. Ca²⁺ influx was shown to be augmented in atherosclerotic artery which might be responsible for abnormal vascular reactivity. However, the mechanism underlying Ca²⁺ influx change in atherosclerotic artery remains undetermined. The purpose of the present study was to examine the effects of LPC on L-type Ca²⁺ current (I_Ca(L)) activity and to elucidate the mechanism of LPC-induced change of I_Ca(L) in rabbit portal vein smooth muscle cells using whole cell patch clamp. Extracellular application of LPC increased I_Ca(L) through whole test potentials, and this effect was readily reversed by washout. Steady state voltage dependency of activation or inactivation properties of I_Ca(L) was not significantly changed by LPC. Staurosporine (100 nM) or chelerythrine (3 µM), which is a potent inhibitor of PKC, significantly decreased basal I_Ca(L), and LPC-induced increase of I_Ca(L) was significantly suppressed in the presence of PKC inhibitors. On the other hand, application of PMA, an activator of PKC, increased basal I_Ca(L) significantly, and LPC-induced enhancement of I_Ca(L) was abolished by pretreatment of the cells with PMA. These findings suggest that LPC increased I_Ca(L) in vascular smooth muscle cells by a pathway that involves PKC, and that LPC-induced increase of I_Ca(L) might be, at least in part, responsible for increased Ca²⁺ influx in atherosclerotic artery.     

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.     

Underlying mechanism of actions of tefluthrin,a pyrethroid insecticide,on voltage-gated ion currents and on action currents in pituitary tumor (GH3) cells and GnRH-secreting (GT1-7) neurons

Tefluthrin is a synthetic pyrethroid and involved in acute neurotoxic effects. How this compound affects ion currents in endocrine or neuroendocrine cells remains unclear. Its effects on membrane ion currents in pituitary tumor (GH₃) cells and in hypothalamic (GT1-7) neurons were investigated. Application of Tef (10 μM) increased the amplitude of voltage-gated Na⁺ current (I_Na), along with a slowing in current inactivation and deactivation in GH₃ cells. The current–voltage relationship of I_Na was shifted to more negative potentials in the presence of this compound. Tef increased I_Na with an EC₅₀ value of 3.2 ± 0.8 μM. It also increased the amplitude of persistent I_Na. Tef reduced the amplitude of L-type Ca²⁺ current. This agent slightly inhibited K⁺ outward current; however, it had no effect on the activity of large-conductance Ca²⁺-activated K⁺ channels. Under cell-attached voltage-clamp recordings, Tef (10 μM) increased amplitude and frequency of spontaneous action currents, along with appearance of oscillatory inward currents. Tef-induced inward currents were suppressed after further application of tetrodotoxin, riluzole or ranolazine. In GT1-7 cells, Tef also increased the amplitude and frequency of action currents. Taken together, the effects of Tef and its structural related pyrethroids on ion currents can contribute to the underlying mechanisms through which they affect endocrine or neuroendocrine function in vivo.