Strophanthidin Induces Apoptosis of Human Lung Adenocarcinoma Cells by Promoting TRAIL-DR5 Signaling.

Strophanthidin (SPTD), one of the cardiac glycosides, is refined from traditional Chinese medicines such as Semen Lepidii and Antiaris toxicaria, and was initially used for the treatment of heart failure disease in clinic. Recently, SPTD has been shown to be a potential anticancer agent, but the underlying mechanism of action is poorly understood. Herein, we explored the molecular mechanism by which SPTD exerts anticancer effects in A549 human lung adenocarcinoma cells by means of mass spectrometry-based quantitative proteomics in combination with bioinformatics analysis. We revealed that SPTD promoted the expression of tumor necrosis factor (TNF)-related apoptosis-inducing ligand receptor 2 (TRAIL-R2, or DR5) in A549 cells to activate caspase 3/6/8, in particular caspase 3. Consequently, the activated caspases elevated the expression level of apoptotic chromatin condensation inducer in the nucleus (ACIN1) and prelamin-A/C (LMNA), ultimately inducing apoptosis via cooperation with the SPTD-induced overexpressed barrier-to-autointegration factor 1 (Banf1). Moreover, the SPTD-induced DEPs interacted with each other to downregulate the p38 MAPK/ERK signaling, contributing to the SPTD inhibition of the growth of A549 cells. Additionally, the downregulation of collagen COL1A5 by SPTD was another anticancer benefit of SPTD through the modulation of the cell microenvironment.

RNA adenosine modifications related to prognosis and immune infiltration in osteosarcoma.

BACKGROUND: RNA adenosine modifications, which are primarily mediated by "writer" enzymes (RMWs), play a key role in epigenetic regulation in various biological processes, including tumorigenesis. However, the expression and prognostic role of these genes in osteosarcoma (OS) remain unclear. METHODS: Univariate and multivariate Cox analyses were used to construct the RMW signature for OS using Target datasets. RMW expression in OS tissue was detected by qPCR analysis. Xcell and GSVA were used to determine the relationship between RMWs and immune infiltration. The DGIdb and CMap databases were used for drug prediction. In vivo and in vitro experiments showed that strophanthidin elicited antitumor activity against OS. RESULTS: A 3-RMW (CSTF2, ADAR and WTAP) prognostic signature in OS was constructed using the Target dataset and verified using GEO datasets and 63 independent OS tissues via qPCR analysis. High-risk OS patients had poor overall survival, and the prognostic signature was an independent prognostic factor for OS. Functional studies showed that tumour-, metabolism-, cell cycle- and immune-related pathways were related to high risk. Next, we found that RMW-derived high-risk patients exhibited increased infiltration of M2 macrophages and cDCs. Furthermore, we predicted the potential drugs for OS using the DGIdb and CMap databases. In vivo and in vitro experiments showed that strophanthidin elicited antitumor activity against OS by repressing cell growth and inducing cell cycle arrest at the G1 phase. CONCLUSION: The 3-RWM-based prognostic signature established in this study is a novel gene signature associated with immune infiltration, and strophanthidin was identified as a candidate therapy for OS by repressing OS cell growth and the cell cycle.

Acaricidal activity of strophanthidin derivatives against Psoroptes cuniculi and their inhibitory effect on Na+-K+-ATPase.

In our previous studies, we found that as the active gradients of Adonis coerulea, cardenolides and cardiac glycosides presented toxicity against mites by inhibiting Na+-K+-ATPase. In this paper, after evaluating the acaricidal activity of the commercial cardiac aglycones/glycosides, serials of novel strophanthidin derivatives were designed and synthesized with an efficient and simple route under mild conditions, and their toxicity against mites, the cytotoxicity and inhibitory effect on Na+-K+-ATP enzyme in PC12 cells were investigated. Results showed among of all compounds, including 9 commercial agent and 32 synthesized strophanthidin derivatives, QXG-1 presented the strongest toxicity against mites with the LC50 value of 320.0 µg/mL. C-19 group of strophanthidin substituted with glycinemethylester would increase the toxicity against mites, and the hydroxyl group at C-5 play the vital role in terms of the toxicity. At the given concentration, QXG-1 displayed the safety against PC12 (10.0 µg/mL) in vitro and mice (3.2 mg/kg) in acute toxicity test, and strong inhibitory effect on Na+-K+-ATPase. It could be used as a promising acaricidal agent. This study lays the foundation to develop of QXG-1 as a relatively safe and alternative acaricidal agent.

Na+-K+-ATPase functions in the developing hippocampus: regional differences in CA1 and CA3 neuronal excitability and role in epileptiform network bursting.

The Na+-K+-ATPase (Na+-K+ pump) is essential for setting resting membrane potential and restoring transmembrane Na+ and K+ gradients after neuronal firing, yet its roles in developing neurons are not well understood. This study examined the contribution of the Na+-K+ pump to resting membrane potential and membrane excitability of developing CA1 and CA3 neurons and its role in maintaining synchronous network bursting. Experiments were conducted in postnatal day (P)9 to P13 rat hippocampal slices using whole cell patch-clamp and extracellular field-potential recordings. Blockade of the Na+-K+ pump with strophanthidin caused marked depolarization (23.1 mV) in CA3 neurons but only a modest depolarization (3.3 mV) in CA1 neurons. Regarding other membrane properties, strophanthidin differentially altered the voltage-current responses, input resistance, action-potential threshold and amplitude, rheobase, and input-output relationship in CA3 vs. CA1 neurons. At the network level, strophanthidin stopped synchronous epileptiform bursting in CA3 induced by 0 Mg2+ and 4-aminopyridine. Furthermore, dual whole cell recordings revealed that strophanthidin disrupted the synchrony of CA3 neuronal firing. Finally, strophanthidin reduced spontaneous excitatory postsynaptic current (sEPSC) bursts (i.e., synchronous transmitter release) and transformed them into individual sEPSC events (i.e., nonsynchronous transmitter release). These data suggest that the Na+-K+ pump plays a more profound role in membrane excitability in developing CA3 neurons than in CA1 neurons and that the pump is essential for the maintenance of synchronous network bursting in CA3. Compromised Na+-K+ pump function leads to cessation of ongoing synchronous network activity, by desynchronizing neuronal firing and neurotransmitter release in the CA3 synaptic network. These findings have implications for the regulation of network excitability and seizure generation in the developing brain.NEW & NOTEWORTHY Despite the extensive literature showing the importance of the Na+-K+ pump in various neuronal functions, its roles in the developing brain are not well understood. This study reveals that the Na+-K+ pump differentially regulates the excitability of CA3 and CA1 neurons in the developing hippocampus, and the pump activity is crucial for maintaining network activity. Compromised Na+-K+ pump activity desynchronizes neuronal firing and transmitter release, leading to cessation of ongoing epileptiform network bursting.

Less Is More: a Mutation in the Chemical Defense Pathway of Erysimum cheiranthoides (Brassicaceae) Reduces Total Cardenolide Abundance but Increases Resistance to Insect Herbivores.

Erysimum cheiranthoides L (Brassicaceae; wormseed wallflower) accumulates not only glucosinolates, which are characteristic of the Brassicaceae, but also abundant and diverse cardenolides. These steroid toxins, primarily glycosylated forms of digitoxigenin, cannogenol, and strophanthidin, inhibit the function of essential Na+/K+-ATPases in animal cells. We screened a population of 659 ethylmethanesulfonate-mutagenized E. cheiranthoides plants to identify isolates with altered cardenolide profiles. One mutant line exhibited 66% lower cardenolide content, resulting from greatly decreased cannogenol and strophanthidin glycosides, partially compensated for by increases in digitoxigenin glycosides. This phenotype was likely caused by a single-locus recessive mutation, as evidenced by a wildtype phenotype of F1 plants from a backcross, a 3:1 wildtype:mutant segregation in the F2 generation, and genetic mapping of the altered cardenolide phenotype to one position in the genome. The mutation created a more even cardenolide distribution, decreased the average cardenolide polarity, but did not impact most glucosinolates. Growth of generalist herbivores from two feeding guilds, Myzus persicae Sulzer (Hemiptera: Aphididae; green peach aphid) and Trichoplusia ni Hübner (Lepidoptera: Noctuidae; cabbage looper), was decreased on the mutant line compared to wildtype. Both herbivores accumulated cardenolides in proportion to the plant content, with T. ni accumulating higher total concentrations than M. persicae. Helveticoside, a relatively abundant cardenolide in E. cheiranthoides, was not detected in M. persicae feeding on these plants. Our results support the hypothesis that increased digitoxigenin glycosides provide improved protection against M. persicae and T. ni, despite an overall decrease in cardenolide content of the mutant line.

The cardenolides strophanthidin, digoxigenin and dihydroouabain act as activators of the human RORγ/RORγT receptors.

Two isoforms of a ligand-activated nuclear receptor, RORγ and RORγT, have been implicated in various physiological functions, including energy metabolism, circadian rhythm and immune system development. Using a stably transfected reporter cell line, we screened two chemical libraries and identified three cardenolides (natural, plant-derived pesticides) as activators of RORγ-dependent transcription. These compounds increased G6PC and NPAS2 expression in HepG2 cells, accompanied by increased occupancy of RORγ within the promoters of these genes. Further, strophanthidin, digoxigenin and dihydroouabain upregulated IL17A and IL17F expression and enhanced IL17 secretion in Th17 human lymphocytes. Molecular docking analyses of these compounds to the RORγ LBD showed favorable docking scores, suggesting that cardenolides may act as agonists of the receptor. Thus, our results provide new chemical structures for further development of RORγ-selective modulators with virtual therapeutic potential.

Istaroxime, a potential anticancer drug in prostate cancer, exerts beneficial functional effects in healthy and diseased human myocardium.

The current gold standard for prostate cancer treatment is androgen deprivation therapy and antiandrogenic agents. However, adverse cardiovascular events including heart failure can limit therapeutic use. Istaroxime, which combines Na+-K+-ATPase (NKA) inhibition with sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) stimulation, has recently shown promising anti-neoplastic effects in prostate cancer (PC) models and may also improve cardiac function. Considering the promising anticancer effects of istaroxime, we aimed to assess its functional effects on human myocardium. RESULTS: Istaroxime and strophanthidin elicited dose-dependent positive inotropic effects with a decline in developed force at supraphysiological concentrations in human atrial, nonfailing, and failing ventricular (ToF) myocardium. Diastolic force and RT50% did not change after exposure to both drugs. The maximal developed force in our in-vitro model of heart failure (ToF) was significantly higher after istaroxime administration. Such a difference did not occur in atrial or nonfailing ventricular trabeculae and was not applicable to the diastolic force. MATERIALS AND METHODS: Human atrial and ventricular trabeculae were isolated from nonfailing hearts and hearts of infants with tetralogy of Fallot (ToF), which were used as an in-vitro model of heart failure. The samples were electrically stimulated and treated with increasing concentrations of istaroxime and strophanthidin (10 nM-1 µM). Systolic and diastolic force development and relaxation parameters (RT50%) were analyzed. CONCLUSIONS: Combined NKA inhibition/SERCA2a stimulation increases contractility in atrial, nonfailing, and failing myocardium. Considering that heart failure is a potential side effect of current PC treatments, especially in elderly patients, istaroxime might combine beneficial cardiac and anti-cancer properties.

Characterization of the cardiac glycoside and lipid profiles of Strophanthus kombé Oliv. seeds.

The seeds of Strophanthus kombé Oliv. are known to contain high levels of cardioactive compounds. However, the therapeutic use of Strophanthus in the treatment of cardiopathy requires more detailed knowledge of the compound profile to profit from the full potential of Strophanthus preparations. Therefore, the objective was to characterize the cardenolide profile and lipophilic constituents in S. kombé seeds using methods applicable in routine quality control. Freshly prepared S. kombé seed extracts were analyzed without previous sample clean-up using a novel HPLC-DAD-MSn method. In addition, seed oils were analyzed by GC-MS following derivatization of the lipids. More than 20 cardenolides were tentatively assigned in the seed extracts including strophanthidin, strophanthidol, periplogenin and strophanthidinic acid aglycones, carrying various saccharide moieties. The findings revealed the presence of eight novel cardenolides, which have not been described for S. kombé so far. The occurrence of strophanthidinic acid derivatives was verified by comparison with synthesized strophanthidinic acid-cymaropyranoside. GC-MS characterization of the oils mainly revealed the presence of fatty acids, especially oleic acid and linoleic acid, as well as phytosterols, the latter representing intermediates of cardenolide biosynthesis. In summary, these findings broaden our knowledge on the secondary metabolism of Strophanthus.

The mechanism underlying maintenance of the endocochlear potential by the K+ transport system in fibrocytes of the inner ear.

The endocochlear potential (EP) of +80 mV in the scala media, which is indispensable for audition, is controlled by K+ transport across the lateral cochlear wall. This wall includes two epithelial barriers, the syncytium and the marginal cells. The former contains multiple cell types, such as fibrocytes, which are exposed to perilymph on their basolateral surfaces. The apical surfaces of the marginal cells face endolymph. Between the two barriers lies the intrastrial space (IS), an extracellular space with a low K+ concentration ([K+]) and a potential similar to the EP. This intrastrial potential (ISP) dominates the EP and represents the sum of the diffusion potential elicited by a large K+ gradient across the apical surface of the syncytium and the syncytium's potential, which is slightly positive relative to perilymph. Although a K+ transport system in fibrocytes seems to contribute to the EP, the mechanism remains uncertain. We examined the electrochemical properties of the lateral wall of guinea pigs with electrodes sensitive to potential and K+ while perfusing into the perilymph of the scala tympani blockers of Na+,K+-ATPase, the K+ pump thought to be essential to the system. Inhibiting Na+,K+-ATPase barely affected [K+] in the IS but greatly decreased [K+] within the syncytium, reducing the K+ gradient across its apical surface. The treatment hyperpolarized the syncytium only moderately. Consequently, both the ISP and the EP declined. Fibrocytes evidently use the Na+,K+-ATPase to achieve local K+ transport, maintaining the syncytium's high [K+] that is crucial for the K+ diffusion underlying the positive ISP.

Regulation of central Na+ detection requires the cooperative action of the NaX channel and α1 Isoform of Na+/K+-ATPase in the Na+-sensor neuronal population.

The median preoptic nucleus (MnPO) holds a strategic position in the hypothalamus. It is adjacent to the third ventricle; hence, it can directly access the ionic composition of the CSF. MnPO neurons play a critical role in hydromineral homeostasis regulation by acting as central sensors of extracellular Na(+) concentration ([Na(+)](ext)). The mechanism underlying Na(+) sensing involves the atypical Na(+) channel, Na(X). Here we sought to determine whether Na(+) influx in Na(+) sensors is actively regulated via interaction with other membrane proteins involved in cellular Na(+) homeostasis, such as Na(+)/K(+)-ATPase. The Na(+)/K(+)-ATPase role was investigated using patch-clamp recordings in rat MnPO dissociated neurons. Na(+) current evoked with hypernatriuric solution was diminished in the absence of ATP/GTP, indicating that Na(+)/K(+)-ATPase play a central role in [Na(+)](ext) detection. Specific blockers of α1 and α3 isoforms of Na(+)/K(+)-ATPase, ouabain or strophanthidin, inhibited this Na(+) current. However, strophanthidin, which selectively blocks the α1 isoform, was more effective in blocking Na(+) current, suggesting that the Na(+)/K(+)-ATPase-α1 isoform is specifically involved in [Na(+)](ext) detection. Although strophanthidin did not alter either the membrane resistance or the Na(+) reversal potential, the conductance and the permeability of the Na(X) channel decreased significantly. Our results suggest that Na(+)/K(+)-ATPase interacts with the Na(X) channel and regulates the high [Na(+)](ext)-evoked Na(+) current via influencing the Na(+) influx rate. This study describes a novel intracellular regulatory pathway of [Na(+)](ext) detection in MnPO neurons. The α1 isoform of Na(+)/K(+)-ATPase acts as a direct regulatory partner of the Na(X) channel and influences Na(+) influx via controlling the Na(+) permeability of the channel.

Small molecule ligands for enhanced intracellular delivery of lipid nanoparticle formulations of siRNA.

Gene silencing activity of lipid nanoparticle (LNP) formulations of siRNA requires LNP surface factors promoting cellular uptake. This study aimed to identify small molecules that enhance cellular uptake of LNP siRNA systems, then use them as LNP-associated ligands to improve gene silencing potency. Screening the Canadian Chemical Biology Network molecules for effects on LNP uptake into HeLa cells found that cardiac glycosides like ouabain and strophanthidin caused the highest uptake. Cardiac glycosides stimulate endocytosis on binding to plasma membrane Na(+)/K(+) ATPase found in all mammalian cells, offering the potential to stimulate LNP uptake into various cell types. A PEG-lipid containing strophanthidin at the end of PEG (STR-PEG-lipid) was synthesized and incorporated into LNP. Compared to non-liganded systems, STR-PEG-lipid enhanced LNP uptake in various cell types. Furthermore, this enhanced uptake improved marker gene silencing in vitro. Addition of STR-PEG-lipid to LNP siRNA may have general utility for enhancing gene silencing potency. FROM THE CLINICAL EDITOR: In this study, the authors identified small molecules that enhance cellular uptake of lipid nanoparticle siRNA systems, then used them as LNP-associated ligands to improve gene silencing potency.

Intracellular Na(+) and metabolic modulation of Na/K pump and excitability in the rat suprachiasmatic nucleus neurons.

Na/K pump activity and metabolic rate are both higher during the day in the suprachiasmatic nucleus (SCN) that houses the circadian clock. Here we investigated the role of intracellular Na(+) and energy metabolism in regulating Na/K pump activity and neuronal excitability. Removal of extracellular K(+) to block the Na/K pump excited SCN neurons to fire at higher rates and return to normal K(+) to reactivate the pump produced rebound hyperpolarization to inhibit firing. In the presence of tetrodotoxin to block the action potentials, both zero K(+)-induced depolarization and rebound hyperpolarization were blocked by the cardiac glycoside strophanthidin. Ratiometric Na(+) imaging with a Na(+)-sensitive fluorescent dye indicated saturating accumulation of intracellular Na(+) in response to pump blockade with zero K(+). The Na(+) ionophore monensin also induced Na(+) loading and hyperpolarized the membrane potential, with the hyperpolarizing effect of monensin abolished in zero Na(+) or by pump blockade. Conversely, Na(+) depletion with Na(+)-free pipette solution depolarized membrane potential but retained residual Na/K pump activity. Cyanide inhibition of oxidative phosphorylation blocked the Na/K pump to depolarize resting potential and increase spontaneous firing in most cells, and to raise intracellular Na(+) levels in all cells. Nonetheless, the Na/K pump was incompletely blocked by cyanide but completely blocked by iodoacetate to inhibit glycolysis, indicating the involvement of both oxidative phosphorylation and glycolysis in fueling the Na/K pump. Together, the results indicate the importance of intracellular Na(+) and energy metabolism in regulating Na/K pump activity as well as neuronal excitability in the SCN neurons.

Serotonin-mediated modulation of Na+/K+ pump current in rat hippocampal CA1 pyramidal neurons.

BACKGROUND: The aim of this study was to investigate whether serotonin (5-hydroxytryptamine, 5-HT) can modulate Na+/K+ pump in rat hippocampal CA1 pyramidal neurons. RESULTS: 5-HT (0.1, 1 mM) showed Na+/K+ pump current (Ip) densities of 0.40 ± 0.04, 0.34 ± 0.03 pA/pF contrast to 0.63 ± 0.04 pA/pF of the control of 0.5 mM strophanthidin (Str), demonstrating 5-HT-induced inhibition of Ip in a dose-dependent manner in hippocampal CA1 pyramidal neurons. The effect was partly attenuated by ondasetron, a 5-HT3 receptor (5-HT3R) antagonist, not by WAY100635, a 5-HT1AR antagonist, while 1-(3-Chlorophenyl) biguanide hydrochloride (m-CPBG), a 5-HT3R specific agonist, mimicked the effect of 5-HT on Ip. CONCLUSION: 5-HT inhibits neuronal Na+/K+ pump activity via 5-HT3R in rat hippocampal CA1 pyramidal neurons. This discloses novel mechanisms for the function of 5-HT in learning and memory, which may be a useful target to benefit these patients with cognitive disorder.

The alpha-synuclein 5'untranslated region targeted translation blockers: anti-alpha synuclein efficacy of cardiac glycosides and Posiphen.

Increased brain α-synuclein (SNCA) protein expression resulting from gene duplication and triplication can cause a familial form of Parkinson's disease (PD). Dopaminergic neurons exhibit elevated iron levels that can accelerate toxic SNCA fibril formation. Examinations of human post mortem brain have shown that while mRNA levels for SNCA in PD have been shown to be either unchanged or decreased with respect to healthy controls, higher levels of insoluble protein occurs during PD progression. We show evidence that SNCA can be regulated via the 5'untranslated region (5'UTR) of its transcript, which we modeled to fold into a unique RNA stem loop with a CAGUGN apical loop similar to that encoded in the canonical iron-responsive element (IRE) of L- and H-ferritin mRNAs. The SNCA IRE-like stem loop spans the two exons that encode its 5'UTR, whereas, by contrast, the H-ferritin 5'UTR is encoded by a single first exon. We screened a library of 720 natural products (NPs) for their capacity to inhibit SNCA 5'UTR driven luciferase expression. This screen identified several classes of NPs, including the plant cardiac glycosides, mycophenolic acid (an immunosuppressant and Fe chelator), and, additionally, posiphen was identified to repress SNCA 5'UTR conferred translation. Western blotting confirmed that Posiphen and the cardiac glycoside, strophanthidine, selectively blocked SNCA expression (~1 µM IC(50)) in neural cells. For Posiphen this inhibition was accelerated in the presence of iron, thus providing a known APP-directed lead with potential for use as a SNCA blocker for PD therapy. These are candidate drugs with the potential to limit toxic SNCA expression in the brains of PD patients and animal models in vivo.

Asymmetric control of inspiratory and expiratory phases by excitability in the respiratory network of neonatal mice in vitro.

Rhythmic motor behaviours consist of alternating movements, e.g. swing-stance in stepping, jaw opening and closing during chewing, and inspiration-expiration in breathing, which must be labile in frequency, and in some cases, in the duration of individual phases, to adjust to physiological demands. These movements are the expression of underlying neural circuits whose organization governs the properties of the motor behaviour. To determine if the ability to operate over a broad range of frequencies in respiration is expressed in the rhythm generator, we isolated the kernel of essential respiratory circuits using rhythmically active in vitro slices from neonatal mice. We show respiratory motor output in these slices at very low frequencies (0.008 Hz), well below the typical frequency in vitro (approximately 0.2 Hz) and in most intact normothermic mammals. Across this broad range of frequencies, inspiratory motor output bursts remained remarkably constant in pattern, i.e. duration, peak amplitude and area. The change in frequency was instead attributable to increased interburst interval, and was largely unaffected by removal of fast inhibitory transmission. Modulation of the frequency was primarily achieved by manipulating extracellular potassium, which significantly affects neuronal excitability. When excitability was lowered to slow down, or in some cases stop, spontaneous rhythm, brief stimulation of the respiratory network with a glutamatergic agonist could evoke (rhythmic) motor output. In slices with slow (<0.02 Hz) spontaneous rhythms, evoked motor output could follow a spontaneous burst at short (60 s. We observed during inspiration a large magnitude (approximately 0.6 nA) outward current generated by Na(+)/K(+) ATPase that deactivated in 25-100 ms and thus could contribute to burst termination and the latency of evoked bursts but is unlikely to control the interburst interval. We propose that the respiratory network functions over a broad range of frequencies by engaging distinct mechanisms from those controlling inspiratory duration and pattern that specifically govern the interburst interval.

Different Na+/K+-ATPase signal pathways was involved in the increase of [Ca2+]i induced by strophanthidin in normal and failing isolated guinea pig ventricular myocytes.

AIM: To determine whether different Na+/K+-ATPase signal transduction pathways have positive inotropic effects on normal ventricular myocytes (NC) and failing ventricular myocytes (FC), and are involved in an increase of [Ca2+]i induced by strophanthidin (Str). METHODS: A guinea pig model of congestive heart failure was made by constricting descending aorta. The left ventricular myocytes were enzymatically isolated. The effects of 25 micromol/L Str with different signal-transducing inhibitors on contractility and the calcium transient of NC or FC from guinea pigs were simultaneously assessed and compared with those in the 25 micromol/L Str-only group by a video-based, motion-edge detection system. RESULTS: Str at 1, 10, and 25 micromol/L in NC and Str at 0.1, 1, 10, and 25 micromol/L) in FC elevated the calcium transient amplitude and increased the positive inotropic effects in a concentration-dependent manner, respectively. At the same concentration, the effects of Str were more potent in FC than in NC. In FC, both the mitogen-activated protein kinase (MAPK) and reactive oxygen species (ROS) signal transduction pathway of Na+/K+-ATPase were involved in the increase of the calcium transient induced by Str, but only activation of the MAPK pathway increased the calcium transient in NC. However, only the ROS pathway was involved in positive inotropic effects both in NC and FC. CONCLUSION: The present study suggests that Na+/K+-ATPase signaling pathways involved in the inotropic effects of Str in NC and FC are consistent, and Na+/K+-ATPase signaling pathways involved in the increase of [Ca2+]i by Str in NC and FC are different.

Effects of strophanthidin on intracellular calcium concentration in ventricular myocytes of guinea pig.

Effect of strophanthidin (Str) on intracellular calcium concentration ([Ca2+]i) was investigated on isolated ventricular myocytes of guinea pig. Single ventricular myocytes were obtained by enzymatic dissociation technique. Fluorescent signal of [Ca2+]i was detected with confocal microscopy after incubation of cardiomycytes in Tyrode' s solution with Fluo3-AM. The result showed that Str increased [Ca2+]i in a concentration-dependent manner. The ventricular myocytes began to round-up into a contracture state once the peak level of [Ca2+]i was achieved in the presence of Str (10 micromol L(- 1)), but remained no change in the presence of Str (1 and 100 nmol L(-1)). Tetrodotoxin (TTX), nisodipine, and high concentration of extracellular Ca2+ changed the response of cardiomycytes to Str (1 and 100 nmol L(-1)) , but had no obvious effects on the action of Str (10 micromol L(-1)). The elevation of [Ca2+]i caused by Str at all of the detected concentrations was partially antagonized by rynodine (10 micromol L(-1)) or the removal of Ca2+ from Tyrode's solution. In Na+, K+ -free Tyrode' s solution, the response of cardiomycytes in [Ca2+]i elevation to Str (10 micromol L(-1)) was attenuated, while remained no change to Str (1 and 100 nmol L(-1)). TTX, nisodipine, and high concentration of extracellular Ca2+ changed the response of cardiomycytes to Str at all of the detected concentrations in Na+, K+ -free Tyrode's solution. The study suggests that the elevation of [Ca2+]i by Str at the low (nomomolar) concentrations is partially mediated by the extracellular calcium influx through Ca2+ channel or a "slip mode conductance" of TTX sensitive Na+ channel. While the effect of Str at high (micromolar) concentrations was mainly due to the inhibition of Na+, K+ -ATPase. Directly triggering the release of intracellular Ca2+ from sarcoplasmic reticulum (SR) by Str may be also involved in the mechanism of [Ca2+]i elevation.

On the mechanisms of arrhythmias in the myocardium of mXinalpha-deficient murine left atrial-pulmonary veins.

We have previously shown that left atrial-pulmonary vein tissue (LA-PV) can generate reentrant arrhythmias (atrial fibrillation, AF) in wild-type (mXinalpha+/+) but not in mXinalpha-null (mXinalpha-/-) mice. With the present experiments, we investigated the arrhythmogenic activity and the underlying mechanisms in mXinalpha+/+ vs. mXinalpha-/- LA-PV. Electrical activity and conduction velocity (CV) were recorded in LA-PV by means of a MED64 system. CV was significantly faster in mXinalpha+/+ than in mXinalpha-/- LA-PV and it was increased by 1 muM isoproterenol (ISO). AF could be induced by fast pacing in the mXinalpha+/+ but not in mXinalpha-/- LA-PV where automatic rhythms could occur. ISO increased the incidence of AF in Xinalpha+/+ whereas it increased that of automatic rhythms in mXinalpha-/- LA-PV. In LA-PV with the right atrium attached (RA-LA-PV), automatic rhythms occurred in all preparations. In mXinalpha+/+ RA-LA-PV simultaneously treated with ISO, strophanthidin and atropine, the incidence of the automatic rhythm was about the same, but AF increased significantly. In contrast, in mXinalpha-/- RA-LA-PV under the same condition, the automatic rhythm was markedly enhanced, but still no AF occurred. Conventional microelectrode techniques showed a longer APD(90) and a less negative maximum diastolic potential (MDP) in mXinalpha-/- than mXinalpha+/+ LA-PV tissues. Whole-cell current clamp experiments also showed a less negative MDP in mXinalpha-/- vs. mXinalpha+/+ LA-PV cardiomyocytes. The fact that AF could be induced by fast pacing under several conditions in mXinalpha+/+ but not in mXinalpha-/- LA-PV preparations appears to be due to a slower CV, a prolonged APD(90), a less negative MDP and possibly larger areas of conduction block in mXinalpha-/- myocardial cells. In contrast, the non-impairment of automatic and triggered rhythms in mXinalpha-/- preparations may be due to the fact that the mechanisms underlying these rhythms do not involve cell-to-cell conduction.

Sodium pumps adapt spike bursting to stimulus statistics.

Pump activity is a homeostatic mechanism that maintains ionic gradients. Here we examined whether the slow reduction in excitability induced by sodium-pump activity that has been seen in many neuronal types is also involved in neuronal coding. We took intracellular recordings from a spike-bursting sensory neuron in the leech Hirudo medicinalis in response to naturalistic tactile stimuli with different statistical distributions. We show that regulation of excitability by sodium pumps is necessary for the neuron to make different responses depending on the statistical context of the stimuli. In particular, sodium-pump activity allowed spike-burst sizes and rates to code not for stimulus values per se, but for their ratio with the standard deviation of the stimulus distribution. Modeling further showed that sodium pumps can be a general mechanism of adaptation to statistics on the time scale of 1 min. These results implicate the ubiquitous pump activity in the adaptation of neural codes to statistics.