Essential oil of Sicilian Prangos ferulacea (L.) Lindl. and its major component, ß-ocimen, affect contractility in rat small and large intestine.

ETHNOPHARMACOLOGICAL RELEVANCE: Prangos ferulacea (L.) Lindl is an Apiaceae plant, widely used in traditional medicine. Recently, chemical composition and biological activities of its essential oil (Prangroil) have been reported, but there are no studies on possible effects on intestinal contractility. AIMS OF THE STUDY: We investigated the effects of essential oil Sicilian Prangoil on the contractility of rat small (duodenum) and large (colon) intestine and the related action mechanism. MATERIALS AND METHODS: Responses to Prangoil and to its major component ß-ocimen in intestinal segments were assessed in vitro as changes in isometric tension. RESULTS: Prangoil, induced in duodenum, depending upon doses, contraction and/or muscular relaxation. Instead, in colon Prangoil only reduced the phasic contractions and induced muscular relaxation. ß-ocimen, in both segments, produced only reduction of the spontaneous contractions without affecting basal tone. Prangoil contractile effects were abolished by ω-conotoxin, neural N-type Ca2+ channels blocker, atropine, muscarinic receptor antagonist, neostigmine, acetylcholinesterase (AChE) inhibitor, suggesting that Prangoil-induced contraction would be the result of an increase in neuronal cholinergic activity. Prangoil and ß-ocimen inhibitory effects were unaffected by ω-conotoxin, L-NAME, blocker of the NO synthase, ODQ, soluble guanylate cyclase inhibitor, excluding involvement of neurotransmitter release or NO synthesis in the inhibitory effects. Potassium channel blocker did not affect Prangoil or ß-ocimen inhibitory responses. Prangoil or ß-ocimen inhibited the Ca2+ and high-KCl solution -induced contractions and the Carbachol-induced contractions in calcium free solution. CONCLUSION: Prangoil affects the contractility of small and large intestine in rat, with regional differences, via potentiation of neural cholinergic activity, blockade of L-type voltage-gated calcium channel and reduction of Ca2+ release from the intracellular store. The Prangroil main components, ß-ocimen, contributes to the inhibitory effects.

A Simulation Study on the Pacing and Driving of the Biological Pacemaker.

The research on the biological pacemaker has been very active in recent years. And turning nonautomatic ventricular cells into pacemaking cells is believed to hold the key to making a biological pacemaker. In the study, the inward-rectifier K+ current (I K1) is depressed to induce the automaticity of the ventricular myocyte, and then, the effects of the other membrane ion currents on the automaticity are analyzed. It is discovered that the L-type calcium current (I CaL) plays a major part in the rapid depolarization of the action potential (AP). A small enough I CaL would lead to the failure of the automaticity of the ventricular myocyte. Meanwhile, the background sodium current (I bNa), the background calcium current (I bCa), and the Na+/Ca2+ exchanger current (I NaCa) contribute significantly to the slow depolarization, indicating that these currents are the main supplementary power of the pacing induced by depressing I K1, while in the 2D simulation, we find that the weak electrical coupling plays a more important role in the driving of a biological pacemaker.

Hypotensive effect of Eugenia dysenterica leaf extract is primarily related to its vascular action: The possible underlying mechanisms.

ETHNOPHARMACOLOGICAL RELEVANCE: Eugenia dysenterica (ED) leaves are used in Brazil to treat cardiac diseases; however, there are no scientific data describing the effects of this species on cardiac activity. AIM OF THE STUDY: To investigate the effect of ED aqueous leaf extract (EDLE) on hear rate (HR) and mean arterial pressure (MAP) of anaesthetised rats and its underlying mechanism of action. MATERIAL AND METHODS: EDLE was analysed, and its proanthocyanidin composition was determined. After performing dose-effect curves for EDLE on HR and MAP, EDLE-induced hypotension was evaluated before and after atropine (AT), L-N(ω)-nitro-L-arginine methyl ester (L-NAME), hexamethonium (HXT), indomethacin (IND), carbenoxolone (CBX), or nifedipine (NFD) administration. The effect of proanthocyanidin-depleted extract (EDLE/P-) was also determined and compared to that of the EDLE with proanthocyanidins. RESULTS: EDLE decreased the MAP in a dose-dependent manner; HR was decreased only with the highest and most toxic dose. Only CBX and NFD decreased EDLE-induced hypotension. Five polymeric series of proanthocyanidins were identified, which were mainly constituted by procyanidin and prodelphinidin units with B-type linkage and up to 12 flavan-3-ol units. EDLE/P- induced hypotension did not differ from that induced by EDLE. CONCLUSIONS: The cardiovascular effects of EDLE were primarily related to its vascular action. EDLE-induced hypotensive effect appeared to involve L-type calcium channel blockage as well as myoendothelial gap junction signalling. The higher molecular weight proanthocyanidins from EDLE are unlikely to contribute to its cardiovascular effect.

Coordinated calcium signalling in cochlear sensory and non-sensory cells refines afferent innervation of outer hair cells.

Outer hair cells (OHCs) are highly specialized sensory cells conferring the fine-tuning and high sensitivity of the mammalian cochlea to acoustic stimuli. Here, by genetically manipulating spontaneous Ca2+ signalling in mice in vivo, through a period of early postnatal development, we find that the refinement of OHC afferent innervation is regulated by complementary spontaneous Ca2+ signals originating in OHCs and non-sensory cells. OHCs fire spontaneous Ca2+ action potentials during a narrow period of neonatal development. Simultaneously, waves of Ca2+ activity in the non-sensory cells of the greater epithelial ridge cause, via ATP-induced activation of P2X3 receptors, the increase and synchronization of the Ca2+ activity in nearby OHCs. This synchronization is required for the refinement of their immature afferent innervation. In the absence of connexin channels, Ca2+ waves are impaired, leading to a reduction in the number of ribbon synapses and afferent fibres on OHCs. We propose that the correct maturation of the afferent connectivity of OHCs requires experience-independent Ca2+ signals from sensory and non-sensory cells.

Hibiscus acid from Hibiscus sabdariffa (Malvaceae) has a vasorelaxant effect on the rat aorta.

Hibiscus sabdariffa (Malvaceae) is a plant that is widely recognised for its antihypertensive properties; however the constituent(s) responsible for this biological activity are presently unknown. The aim of this study was to identify the potential compounds that are responsible for the vasorelaxant activity of H. sabdariffa. Thereafter, the mechanisms involved in producing the vasorelaxation were investigated. The plant was extracted consecutively with hexane, ethyl acetate and methanol. The methanolic extract was subjected to bioassay-guided fractionation in order to isolate pure compounds that possessed vasorelaxant activity. The vascular effects of the pure compounds were studied on the rat aorta in vitro using myography techniques. Hibiscus acid produced a concentration-dependent relaxation of the rat aorta pre-contracted with either phenylephrine (3 µM) or KCl (60 mM), irrespective of the presence of the endothelium. When the tissue was pre-contracted with phenylephrine, the concentration required to produce 50% relaxation (IC50), was 0.09 ±â€¯0.01 mg/ml. Hibiscus acid had no effect on the phasic contraction induced by phenylephrine in Ca2+-free physiological solution; but it did affect the component of the contraction that is due to Ca2+ influx. In parallel studies, garcinia acid, a diastereoisomer of hibiscus acid, was found to have an almost identical vasorelaxant effect. The vasorelaxant action of both compounds is most likely due to the inhibition of Ca2+ influx via voltage-dependent Ca2+ channels.

Quercetin Attenuates Ethanol-Induced Iron Uptake and Myocardial Injury by Regulating the Angiotensin II-L-Type Calcium Channel.

SCOPE: Increased iron deposition in the myocardium in alcoholics may lead to increased risk of cardiac dysfunction. Quercetin has been demonstrated to quench production of intracellular free iron-induced -OH, but the effect of quercetin in ethanol-induced cardiac damage remains unclear. This study aims to explore whether quercetin attenuates ethanol-induced iron uptake and myocardial injury by regulating angiotensin II-L-type voltage-dependent Ca2+ channel (Ang II-LTCC). METHODS AND RESULTS: Adult male C57BL/6J mice are isocalorically pair-fed either a regular or ethanol-containing Lieber De Carli liquid diets supplemented with either quercetin (100 mg kg-1  bw) or desferrioxamine mesylate (DFO, 100 mg kg-1 bw) for 15 weeks. Quercetin alleviated ethanol-induced histopathological changes, creatine kinase isoenzyme release, Ang II secretion, ROS generation, total cardiac iron, and labile iron level. Ethanol exposure or quercetin intervention fails to regulate traditional iron transporters except LTCC. LTCC is upregulated by ethanol and inhibited by quercetin. In H9C2 cell, LTCC is increased by ethanol (100 mm) and/or Ang II (1 µm) concomitant with iron disorders and oxidative stress. This effect is partially normalized by quercetin (50 µm), nifedipine (LTCC inhibitor, 15 µm), or losartan (Ang II receptor antagonist, 100 µm). CONCLUSION: Alcohol-induced cardiac injury is associated with excessive NTBI uptake mediated by Ang II-LTCC activation which may be mediated by quercetin against ethanol cardiotoxicity.

Nimodipine, an L-type calcium channel blocker attenuates mitochondrial dysfunctions to protect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism in mice.

Parkinson's disease (PD), the most common progressive neurodegenerative movement disorder, results from loss of dopaminergic neurons of substantia nigra pars compacta. These neurons exhibit Cav1.3 channel-dependent pacemaking activity. Epidemiological studies suggest reduced risk for PD in population under long-term antihypertensive therapy with L-type calcium channel antagonists. These prompted us to investigate nimodipine, an L-type calcium channel blocker for neuroprotective effect in cellular and animal models of PD. Nimodipine (0.1-10 µM) significantly attenuated 1-methyl-4-phenyl pyridinium ion-induced loss in mitochondrial morphology, mitochondrial membrane potential and increases in intracellular calcium levels in SH-SY5Y neuroblastoma cell line as measured respectively employing Mitotracker green staining, TMRM, and Fura-2 fluorescence, but only a feeble neuroprotective effect was observed in MTT assay. Nimodipine dose-dependently reduced 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonian syndromes (akinesia and catalepsy) and loss in swimming ability in Balb/c mice. It attenuated MPTP-induced loss of dopaminergic tyrosine hydroxylase positive neurons in substantia nigra, improved mitochondrial oxygen consumption and inhibited reactive oxygen species production in the striatal mitochondria measured using dichlorodihydrofluorescein fluorescence, but failed to block striatal dopamine depletion. These results point to an involvement of L-type calcium channels in MPTP-induced dopaminergic neuronal death in experimental parkinsonism and more importantly provide evidences for nimodipine to improve mitochondrial integrity and function.

Electrophysiological effect and the gating mechanism of astragaloside IV on L-type Ca(2+) channels of guinea-pig ventricular myocytes.

Astragaloside IV (AS-IV) is one of the main active ingredients of Astragalus membranaceus. This study is aimed to investigate AS-IV׳s effects on Ca(2+) channel activity of single cardiomyocytes and single Ca(2+) channels. Whole-cell Ca(2+) currents in freshly dissociated cardiomyocytes were measured using the whole-cell patch-clamp technique. Single Ca(2+) channel currents were examined in cell-attached patches and inside-out patches. In the whole-cell recording, AS-IV reduced the amplitude of L-type Ca(2+) currents (ICaL) in a concentration-dependent manner. Although AS-IV did not alter the steady-state activation curves, the voltage dependence of the current inactivation curves was negatively shifted by AS-IV in a concentration dependent manner. Consistent with the results of the whole-cell recording, in the inside-out configuration the ensemble average of single Ba(2+) current via L-type Ca(2+) channel was dose-dependently reduced by AS-IV. The reduction of unitary Ba(2+) current at 0.1 or 1 µM AS-IV was accounted for a decrease in the channel activity (NPo). In addition to the decrease in NPo, there was a reduction of Po without a change in channel number or an apparent change in single channel current. Furthermore, we found that the open-closed kinetics of the channel were affected by AS-IV. AS-IV induced the shift of L-type Ca(2+) channels from either brief openings (mode 1) or long-lasting openings (mode 2) to no active opening (mode 0). Our results suggest that AS-IV blocks the currents through Ca(2+) channels in guinea-pig ventricular myocytes by affecting the open-closed kinetics of L-type Ca(2+) channels to inhibit the channel activities. This study could provide theoretical basis for the drug exploiting of the monomer of Astragalus membranaceus.

Vasorelaxation induced by dodoneine is mediated by calcium channels blockade and carbonic anhydrase inhibition on vascular smooth muscle cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Dodoneine (Ddn) is one of the active compounds identified from Agelanthus dodoneifolius (DC.) Polhill and Wiens, a medicinal plant used in traditional medicine for the treatment of hypertension. This dihydropyranone exerts hypotensive and vasorelaxant effects on rats, and two molecular targets have been characterized: the carbonic anhydrase and the L-type calcium channel in cardiomyocytes with biochemical and electrophysiological techniques, respectively. To further evaluate the involvement of these two molecular targets in vasorelaxation, the effect of Ddn on rat vascular smooth muscle was investigated. MATERIAL AND METHODS: The effects of Ddn on L-type calcium current and on resting membrane potential were characterized in A7r5 cell line using the whole-cell patch-clamp configuration. The molecular identities of carbonic anhydrase isozymes in smooth muscle cells were examined with RT-PCR. Vascular response was measured on rat aortic rings in an organ bath apparatus and the effect of Ddn on intracellular pH was determined by flow cytometry using the pH-sensitive fluorescent probe BCECF-AM [2,7-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein, Acetoxymethyl Ester]. RESULTS: 100µM Ddn reduced calcium current density of about 30%. In addition, carbonic anhydrase II, III, XIII and XIV were shown to be expressed in rat aorta and inhibited in smooth muscle cells by Ddn. This inhibition resulted in a rise in pHi of about 0.31, leading to KCa channel activation, thereby inducing membrane hyperpolarization and vasorelaxation. The results of vascular reactivity experiments obtained with pharmacological tools acting on the L-type calcium current and carbonic anhydrase suggest that Ddn produces its vasorelaxant effect via the inhibition of these two molecular targets. CONCLUSION: This study demonstrates that Ddn induced vasorelaxation by targeting two proteins involved in the modulation of excitation-contraction coupling: L-type calcium channels and carbonic anhydrase.

Emodin accentuates atrial natriuretic peptide secretion in cardiac atria.

Emodin, an active anthraquinone constituent isolated from the rhubarb, a traditional Chinese herbal medicine which is widely used in clinical treatment, has cardiovascular protective properties. However, it remains unclear whether the cardiovascular protective actions of emodin are related to an activation of cardiac natriuretic hormone secretion. The purpose of the present study was to explore the effect of emodin on the secretion of ANP, a member of the family of cardiac natriuretic hormones, and its mechanisms involved. Experiments were performed in isolated perfused beating rabbit atria allowing measurement of ANP secretion, atrial pulse pressure, and stroke volume. Emodin increased ANP secretion concomitantly with a decrease in atrial pulse pressure and stroke volume in a concentration-dependent manner. These effects were reversible. Inhibition of K(+) channels with tetraethylammonium and glibenclamide attenuated the emodin-induced changes in ANP secretion and atrial dynamics. Furthermore, the emodin-induced changes in ANP secretion and atrial dynamics were attenuated by inhibition of L-type Ca(2+) channels with nifedipine. Atropine, methoctramine, tertiapin-Q, and pertussis toxin had no significant effect on the emodin-induced changes in ANP secretion and mechanical dynamics. The present study demonstrates that emodin increases ANP secretion via inhibition of L-type Ca(2+) channels through an activation of K(+)ATP channel in isolated beating rabbit atria. The results also provide a rationale for the use of emodin in the treatment of impairment of the regulation of the cardiovascular homeostasis.

Dominant frequency increase rate predicts transition from paroxysmal to long-term persistent atrial fibrillation.

BACKGROUND: Little is known about the mechanisms underlying the transition from paroxysmal to persistent atrial fibrillation (AF). In an ovine model of long-standing persistent AF we tested the hypothesis that the rate of electric and structural remodeling, assessed by dominant frequency (DF) changes, determines the time at which AF becomes persistent. METHODS AND RESULTS: Self-sustained AF was induced by atrial tachypacing. Seven sheep were euthanized 11.5±2.3 days after the transition to persistent AF and without reversal to sinus rhythm; 7 sheep were euthanized after 341.3±16.7 days of long-standing persistent AF. Seven sham-operated animals were in sinus rhythm for 1 year. DF was monitored continuously in each group. Real-time polymerase chain reaction, Western blotting, patch clamping, and histological analyses were used to determine the changes in functional ion channel expression and structural remodeling. Atrial dilatation, mitral valve regurgitation, myocyte hypertrophy, and atrial fibrosis occurred progressively and became statistically significant after the transition to persistent AF, with no evidence for left ventricular dysfunction. DF increased progressively during the paroxysmal-to-persistent AF transition and stabilized when AF became persistent. Importantly, the rate of DF increase correlated strongly with the time to persistent AF. Significant action potential duration abbreviation, secondary to functional ion channel protein expression changes (CaV1.2, NaV1.5, and KV4.2 decrease; Kir2.3 increase), was already present at the transition and persisted for 1 year of follow up. CONCLUSIONS: In the sheep model of long-standing persistent AF, the rate of DF increase predicts the time at which AF stabilizes and becomes persistent, reflecting changes in action potential duration and densities of sodium, L-type calcium, and inward rectifier currents.

Relaxant effect of 1-butanol fraction from Elaeagnus pungens leaf through inhibiting L-type Ca2+ channel on guinea pig tracheal smooth muscle.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaf of Elaeagnus pungens thunb. (Family Elaeagnaceae) has been documented as an effective herb for the treatment of asthma and chronic bronchitis in traditional Chinese medicine. In the past years, only a few of preliminary studies reported the chemical constituents and pharmacology effects of the herb, but their action on the tracheal relaxation has not been investigated. AIM OF THE STUDY: To investigate the relaxing effect and mechanism of the extracts from Elaeagnus pungens leaves on guinea pig tracheal smooth muscle and bronchi smooth muscle cells. MATERIALS AND METHODS: Four fractions of different polarities from Elaeagnus pungens leaves were tested to the tracheal strips on the resting tension or pre-contracted by histamine (20 µM) and acetylcholine (20 µM). Inhibitory effects of the 1-butanol fraction (400mg/ml) on cumulative histamine and acetylcholine (0.2-20 µM) induced contraction were measured. In order to determine the mediators on the 1-butanol fraction effect, the relaxing effect of the 1-butanol fraction was evaluated in the absence and presence of ß-adrenoceptor antagonists (1 µM propranolol), K(+) channels-blockers (4-aminopyridine (2mM), tetraethylammonium chloride (5mM) or glibenclamide (10 µM)), the cyclooxygenase inhibitor (indomethacin, 10 µM), nitric oxide synthase inhibitor (Nω-nitro-L-arginine methyl ester, 100 µM) or L-type Ca(2+) channel inhibitor (nifedipine, 1 µM). Moreover, [Ca(2+)]i in bronchi smooth muscle cells was analyzed by measuring the fluorescence intensity with confocal system. RESULTS: 1-Butanol fraction induced the highest relaxant effect among four fractions of different polarities from Elaeagnus pungens leaves, and significantly relaxed the tracheal strip in the concentration-dependent manner on the resting tension and pre-contracted by histamine phosphate and acetylcholine. It also produced an unparallel rightward shift of the cumulative concentration-response curve of histamine or acetylcholine. Furthermore, the relaxant effect of 1-butanol fraction was not affected by propranolol, glibenclamide, tetraethylammonium chloride, 4-aminopyridine, indomethacin and Nω-nitro-L-arginine methyl ester. However, 1-butanol fraction-induced relaxation decreased after adding nifedipine. It also concentration-dependently inhibited CaCl2-induced contraction in the Ca(2+)-free, 60mM K(+)-containing solution. Additionally, [Ca(2+)]i in the BSMCs significantly reduced after administration of the 1-butanol fraction. CONCLUSIONS: The 1-butanol fraction from Elaeagnus pungens leaves resulted in a relaxation in the non-precontracted and pre-contracted tracheal strips. The relaxant effect was not related to K(+) channels, NO, cGMP or ß-adrenoceptors, but related to the inhibition of Ca(2+) influx through L-type Ca(2+) channels.

Magnesium sulfate provides neuroprotection in lipopolysaccharide-activated primary microglia by inhibiting NF-κB pathway.

BACKGROUND: Magnesium sulfate has been used as an anticonvulsant in severe preeclamptic or eclamptic women prior to surgical trauma, but its effects on neuroinflammation is not well defined. In the present study, we investigated the neuroprotective effects of magnesium sulfate in lipopolysaccharide (LPS)-induced microglia and explored the underlying mechanism. MATERIALS AND METHODS: Microglia was incubated with LPS in the presence or absence of various concentrations of magnesium sulfate, or L-type calcium channel activator BAY-K8644. The levels of inflammatory mediators, such as nitric oxide, prostaglandin E2, interleukin 1ß, and tumor necrosis factor α, were measured using enzyme-linked immunosorbent assay. The expression of inducible nitric oxide synthase mRNA was detected by reverse-transcription polymerase chain reaction. Nuclear factor κB (NF-κB) activity in the nuclear extract of microglia was detected by NF-κB p50/p65 transcription factor assay kit. RESULTS: Magnesium sulfate at 5 and 10 mmol/L significantly inhibited the release of nitric oxide, prostaglandin E2, interleukin 1ß, and tumor necrosis factor α, and the expression of inducible nitric oxide synthase mRNA in LPS-activated microglia. Furthermore, magnesium sulfate inhibited the translocation of NF-κB from the cytoplasm to the nucleus in a dose-dependent manner. Notably, these effects were significantly reversed by L-type calcium channel activator BAY-K8644. CONCLUSIONS: Magnesium sulfate protects microglia against LPS-induced release of inflammatory mediators, and these effects may be mediated by inhibiting L-type calcium channels and NF-κB signaling.

I(CaL) inhibition prevents arrhythmogenic Ca(2+) waves caused by abnormal Ca(2+) sensitivity of RyR or SR Ca(2+) accumulation.

AIMS: Arrhythmogenic Ca(2+) waves result from uncontrolled Ca(2+) release from the sarcoplasmic reticulum (SR) that occurs with increased Ca(2+) sensitivity of the ryanodine receptor (RyR) or excessive Ca(2+) accumulation during ß-adrenergic stimulation. We hypothesized that inhibition of the L-type Ca(2+) current (I(CaL)) could prevent such Ca(2+) waves in both situations. METHODS AND RESULTS: Ca(2+) waves were induced in mouse left ventricular cardiomyocytes by isoproterenol combined with caffeine to increase RyR Ca(2+) sensitivity. I(CaL) inhibition by verapamil (0.5 µM) reduced Ca(2+) wave probability in cardiomyocytes during electrostimulation, and during a 10 s rest period after ceasing stimulation. A separate type of Ca(2+) release events occurred during the decay phase of the Ca(2+) transient and was not prevented by verapamil. Verapamil decreased Ca(2+) spark frequency, but not in permeabilized cells, indicating that this was not due to direct effects on RyR. The antiarrhythmic effect of verapamil was due to reduced SR Ca(2+) content following I(CaL) inhibition. Computational modelling supported that the level of I(CaL) inhibition obtained experimentally was sufficient to reduce the SR Ca(2+) content. Ca(2+) wave prevention through reduced SR Ca(2+) content was also effective in heterozygous ankyrin B knockout mice with excessive SR Ca(2+) accumulation during ß-adrenergic stimulation. CONCLUSION: I(CaL) inhibition prevents diastolic Ca(2+) waves caused by increased Ca(2+) sensitivity of RyR or excessive SR Ca(2+) accumulation during ß-adrenergic stimulation. In contrast, unstimulated early Ca(2+) release during the decay of the Ca(2+) transient is not prevented, and merits further study to understand the full antiarrhythmic potential of I(CaL) inhibition.

In vivo suppression of microRNA-24 prevents the transition toward decompensated hypertrophy in aortic-constricted mice.

RATIONALE: During the transition from compensated hypertrophy to heart failure, the signaling between L-type Ca(2+) channels in the cell membrane/T-tubules and ryanodine receptors in the sarcoplasmic reticulum becomes defective, partially because of the decreased expression of a T-tubule-sarcoplasmic reticulum anchoring protein, junctophilin-2. MicroRNA (miR)-24, a junctophilin-2 suppressing miR, is upregulated in hypertrophied and failing cardiomyocytes. OBJECTIVE: To test whether miR-24 suppression can protect the structural and functional integrity of L-type Ca(2+) channel-ryanodine receptor signaling in hypertrophied cardiomyocytes. METHODS AND RESULTS: In vivo silencing of miR-24 by a specific antagomir in an aorta-constricted mouse model effectively prevented the degradation of heart contraction, but not ventricular hypertrophy. Electrophysiology and confocal imaging studies showed that antagomir treatment prevented the decreases in L-type Ca(2+) channel-ryanodine receptor signaling fidelity/efficiency and whole-cell Ca(2+) transients. Further studies showed that antagomir treatment stabilized junctophilin-2 expression and protected the ultrastructure of T-tubule-sarcoplasmic reticulum junctions from disruption. CONCLUSIONS: MiR-24 suppression prevented the transition from compensated hypertrophy to decompensated hypertrophy, providing a potential strategy for early treatment against heart failure.

Ca(V)1.3-driven SK channel activation regulates pacemaking and spike frequency adaptation in mouse chromaffin cells.

Mouse chromaffin cells (MCCs) fire spontaneous action potentials (APs) at rest. Ca(v)1.3 L-type calcium channels sustain the pacemaker current, and their loss results in depolarized resting potentials (V(rest)), spike broadening, and remarkable switches into depolarization block after BayK 8644 application. A functional coupling between Ca(v)1.3 and BK channels has been reported but cannot fully account for the aforementioned observations. Here, using Ca(v)1.3(-/-) mice, we investigated the role of Ca(v)1.3 on SK channel activation and how this functional coupling affects the firing patterns induced by sustained current injections. MCCs express SK1-3 channels whose tonic currents are responsible for the slow irregular firing observed at rest. Percentage of frequency increase induced by apamin was found inversely correlated to basal firing frequency. Upon stimulation, MCCs build-up Ca(v)1.3-dependent SK currents during the interspike intervals that lead to a notable degree of spike frequency adaptation (SFA). The major contribution of Ca(v)1.3 to the subthreshold Ca(2+) charge during an AP-train rather than a specific molecular coupling to SK channels accounts for the reduced SFA of Ca(v)1.3(-/-) MCCs. Low adaptation ratios due to reduced SK activation associated with Ca(v)1.3 deficiency prevent the efficient recovery of Na(V) channels from inactivation. This promotes a rapid decline of AP amplitudes and facilitates early onset of depolarization block following prolonged stimulation. Thus, besides serving as pacemaker, Ca(v)1.3 slows down MCC firing by activating SK channels that maintain Na(V) channel availability high enough to preserve stable AP waveforms, even upon high-frequency stimulation of chromaffin cells during stress responses.

The effect of ligustrazine on L-type calcium current, calcium transient and contractility in rabbit ventricular myocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Ligustrazine, the biologically active ingredient isolated from a popular Chinese medicinal plant, Ligusticum chuanxiong Hort. (Umbelliferae), has been used effectively to treat ischemic heart diseases, cerebrovascular and thrombotic vascular diseases since the 1970s. MATERIALS AND METHODS: At present, the effect of ligustrazine on L-type calcium current (I(Ca-L)) of ventricular myocytes remains controversial. In this study, we use the whole-cell patch-clamp techniques and video-based edge detection and dual excitation fluorescence photomultiplier systems to study the effects of ligustrazine on I(Ca-L), and calcium transient and contractility in rabbit ventricular myocytes in the absence and presence of isoprenaline (ISO). RESULTS: Ligustrazine (5 µM) in low concentration did not affect I(Ca-L) (P>0.05), higher concentrations of this drug (10, 20, 40, 80 µM) inhibited I(Ca-L) in a concentration-dependent manner and reduced I(Ca-L) by 9.6 ± 2.9%, 21.0 ± 4.3%, 33.9 ± 4.3%, and 51.6 ± 7.3%, respectively. Under normal conditions, ligustrazine (40 µΜ) reduced baseline of fura-2 fluorescence intensities (FFI, 340/380 ratio), namely diastolic calcium concentration, changes in FFI (ΔFFI, 340/380 ratio) and maximal velocity of Ca(2+) rise and decay (340/380 ratio/ms) by 6.3%, 26.1%, 25.2%, and 26.5%, and decreased sarcomere peak shorting (PS) and maximal velocity of shorting and relengthening by 36.4%, 31.9%, and 25.0%, respectively. Similarly, ligustrazine (40 µM) reduced baseline FFI, ΔFFI, and maximal velocity of Ca(2+) rise and decay by 14.1%, 51.1%, 35.2%, and 41.1%, and reduced sarcomere PS and maximal velocity of shorting and relengthening by 38.6%, 50.0% and 39.1%, respectively, in the presence of ISO. CONCLUSIONS: Ligustrazine not only significantly inhibits I(Ca-L) in a concentration-dependent manner but also suppressed calcium transient and contraction in the absence and presence of ISO.

Effects of Leonurine on L-type calcium channel in rat ventricular myocytes.

Leonurine (Leo) is a special alkaloid principle of Herba leonuri that has recently been suggested to improve cardiovascular functions. To date, there is no direct ionic evidence of Leo on regulating calcium channels in the heart. In the present study, we examined the effects of Leo on action potentials and membrane currents recorded from isolated rat ventricular myocytes with the whole-cell patch clamp technique. Leo 100 µM shortened the action potential duration in a dose-dependent manner. Leo up to 200 µM had no significant effect on the Na+ current (INa) and K+ current (IK). However, Leo depressed the L-type Ca2+ current (ICa,L). In the presence of 20 and 100 µM Leo, the current density was decreased and the voltage at half maximal inactivation V0.5 shift to more negative potential. The recovery time constant was also delayed. In addition, the transcription and protein expression levels of L-type calcium channel (Cav1.2) in primary cultured neonatal myocytes from Sprague-Dawley rats were reduced by Leo treatment in a dose-dependent fashion as assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assays. We conclude that Leo inhibits L-type calcium channels in cardiomyocytes.

Dietary nitrate increases tetanic [Ca2+]i and contractile force in mouse fast-twitch muscle.

Dietary inorganic nitrate has profound effects on health and physiological responses to exercise. Here, we examined if nitrate, in doses readily achievable via a normal diet, could improve Ca(2+) handling and contractile function using fast- and slow-twitch skeletal muscles from C57bl/6 male mice given 1 mm sodium nitrate in water for 7 days. Age matched controls were provided water without added nitrate. In fast-twitch muscle fibres dissected from nitrate treated mice, myoplasmic free [Ca(2+)] was significantly greater than in Control fibres at stimulation frequencies from 20 to 150 Hz, which resulted in a major increase in contractile force at ≤ 50 Hz. At 100 Hz stimulation, the rate of force development was ∼35% faster in the nitrate group. These changes in nitrate treated mice were accompanied by increased expression of the Ca(2+) handling proteins calsequestrin 1 and the dihydropyridine receptor. No changes in force or calsequestrin 1 and dihydropyridine receptor expression were measured in slow-twitch muscles. In conclusion, these results show a striking effect of nitrate supplementation on intracellular Ca(2+) handling in fast-twitch muscle resulting in increased force production. A new mechanism is revealed by which nitrate can exert effects on muscle function with applications to performance and a potential therapeutic role in conditions with muscle weakness.

Vasodilatory effects of ethanol extract of Radix Paeoniae Rubra and its mechanism of action in the rat aorta.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Paeoniae Rubra (RPR) is an important traditional Chinese medicine (TCM) commonly used in clinic for a long history in China. RPR is the radix of either Paeonia lactiflora Pall. or Paeonia veitchii Lynch. RPR has a wide variety of pharmacological actions such as anti-thrombus, anti-coagulation, and anti-atherosclerotic properties, protecting heart and liver. However, the mechanisms involved are to be defined. AIM OF THE STUDY: The aim of the present study was to define the effect of Paeonia lactiflora Pall. extracts on vascular tension and responsible mechanisms in rat thoracic aortic rings. MATERIALS AND METHODS: Ethanol extract of Paeonia lactiflora Pall. (EPL) was examined for their vascular relaxant effects in isolated phenylephrine-precontracted rat thoracic aorta. RESULTS: EPL induced relaxation of the phenylephrine-precontracted aortic rings in a concentration-dependent manner. Vascular relaxation induced by EPL was significantly inhibited by removal of the endothelium or pretreatment of the rings with N(G)-nitro-L-arginine methylester (L-NAME) or 1H-[1,2,4]-oxadiazolo-[4,3-α]-quinoxalin-1-one (ODQ). Extracellular Ca²âº depletion or diltiazem significantly attenuated EPL-induced vasorelaxation. Modulators of the store-operated Ca²âº entry (SOCE), thapsigargin, 2-aminoethyl diphenylborinate and Gd³âº, and an inhibitor of Akt, wortmannin, markedly attenuated the EPL-induced vasorelaxation. Further, the EPL-induced vasorelaxation was significantly attenuated by pretreatment with tetraethylammonium, a non-selective K(Ca) channels blocker, or glibenclamide, an ATP-sensitive K⁺ channels inhibitor, respectively. Inhibition of cyclooxygenases with indomethacin, and adrenergic and muscarinic receptors blockade had no effects on the EPL-induced vasorelaxation. CONCLUSIONS: The present study suggests that EPL relaxes vascular smooth muscle via endothelium-dependent and Akt- and SOCE-eNOS-cGMP-mediated pathways through activation of both K(Ca) and K(ATP) channels and inhibition of L-type Ca²âº channels.