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.

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.

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.

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.

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.

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.

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.

Mechanistic insight into the functional and toxic effects of Strophanthidin in the failing human myocardium.

BACKGROUND: Cardiac glycosides are characterized by a narrow therapeutic range with Ca2+-overload and arrhythmias occurring at higher concentrations. Data on cardiac glycosides in isolated failing human myocardium are scarce and the frequency-dependent actions and toxicity of Strophanthidin have not yet been characterized. AIMS: To determine inotropic responses and toxicity of Strophanthidin in failing human myocardium. METHODS AND RESULTS: Experiments were performed in trabeculae from 64 end-stage failing hearts. Developed force, and intracellular [Ca2+]i and [Na+]i were recorded with Strophanthidin (0.01 to 1 micromol/L; 37 degrees C, 1 Hz) and compared to interventions with distinct mechanisms of action (elevated [Ca2+]o, Isoproterenol, and EMD57033). The effects of Strophanthidin on force-frequency behaviour were also assessed. Strophanthidin exerted concentration-dependent positive inotropic effects. These were paralleled by increases in intracellular [Na+] as well as increasing [Ca2+]i-transients and SR-Ca2+-load. At high concentrations (>0.5 micromol/L), Strophanthidin caused afterglimmers and aftercontractions, with declining developed force despite further increasing [Ca2+]i-transients. The force-frequency-relationship and diastolic function at higher pacing rates was worsened by Strophanthidin in a concentration-dependent manner. CONCLUSIONS: Strophanthidin toxicity was dependent on concentration, calcium load, beating rate and beta-adrenergic receptor activation. Our data support the view that low doses, heart rate control and additional beta-adrenergic receptor blockade are essential in the use of cardiac glycosides in heart failure.

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.

Rotenone potentiates NMDA currents in substantia nigra dopamine neurons.

Rotenone is a pesticide that produces a rodent model of Parkinson's disease. Although much evidence suggests that oxidative stress mediates the toxicity of rotenone on dopamine neurons, rotenone can also potentiate glutamate excitotoxicity. We used whole-cell patch pipettes to investigate actions of rotenone on currents evoked by N-methyl-d-aspartate (NMDA) in dopamine neurons in slices of rat midbrain. After superfusing the slice for 20-30 min, rotenone (100 nM) caused a 162% increase in the average amplitude of inward current evoked by 30 microM NMDA. This effect of rotenone was mimicked by the sodium pump inhibitor strophanthidin (10 microM) and was abolished when pipettes contained an ATP regeneration solution. Although strophanthidin also significantly increased the amplitude of inward currents evoked by (+/-)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; 10 microM), rotenone failed to potentiate AMPA currents. Because rotenone potentiated NMDA- but not AMPA-dependent currents, this suggests that rotenone acts selectively to augment NMDA receptor function. Furthermore, the failure of rotenone to mimic strophanthidin suggests that rotenone does not inhibit sodium pump activity. Our results suggest that an excitotoxic mechanism might contribute to rotenone neurotoxicity.

The inotropic effect of cardioactive glycosides in ventricular myocytes requires Na+-Ca2+ exchanger function.

Glycoside-induced cardiac inotropy has traditionally been attributed to direct Na(+)-K(+)-ATPase inhibition, causing increased intracellular [Na(+)] and consequent Ca(2+) gain via the Na(+)-Ca(2+) exchanger (NCX). However, recent studies suggested alternative mechanisms of glycoside-induced inotropy: (1) direct activation of sarcoplasmic reticulum Ca(2+) release channels (ryanodine receptors; RyRs); (2) increased Ca(2+) selectivity of Na(+) channels (slip-mode conductance); and (3) other signal transduction pathways. None of these proposed mechanisms requires NCX or an altered [Na(+)] gradient. Here we tested the ability of ouabain (OUA, 3 microm), digoxin (DIG, 20 microm) or acetylstrophanthidin (ACS, 4 microm) to alter Ca(2+) transients in completely Na(+)-free conditions in intact ferret and cat ventricular myocytes. We also tested whether OUA directly activates RyRs in permeabilized cat myocytes (measuring Ca(2+) sparks by confocal microscopy). In intact ferret myocytes (stimulated at 0.2 Hz), DIG and ACS enhanced Ca(2+) transients and cell shortening during twitches, as expected. However, prior depletion of [Na(+)](i) (in Na(+)-free, Ca(2+)-free solution) and in Na(+)-free solution (replaced by Li(+)) the inotropic effects of DIG and ACS were completely prevented. In voltage-clamped cat myocytes, OUA increased Ca(2+) transients by 48 +/- 4% but OUA had no effect in Na(+)-depleted cells (replaced by N-methyl-d-glucamine). In permeabilized cat myocytes, OUA did not change Ca(2+) spark frequency, amplitude or spatial spread (although spark duration was slightly prolonged). We conclude that the acute inotropic effects of DIG, ACS and OUA (and the effects on RyRs) depend on the presence of Na(+) and a functional NCX in ferret and cat myocytes (rather than alternate Na(+)-independent mechanisms).

Effects of sodium pump activity on spontaneous firing in neurons of the rat suprachiasmatic nucleus.

Cell-attached and whole cell recording techniques were used to study the effects of electrogenic sodium pump on the excitability of rat suprachiasmatic nucleus (SCN) neurons. Blocking the sodium pump with the cardiac steroid strophanthidin or zero K+ increased the spontaneous firing of SCN neurons to different degrees with different recording modes, whereas turning the sodium pump into a nonselective cation channel with the marine toxin palytoxin invariably increased the spontaneous firing to the point of total blockade. Current-clamp recordings indicated that strophanthidin increased the rate of membrane depolarization and reduced the peak afterhyperpolarization potential (AHP), whereas zero K+ also increased the rate of depolarization, but enhanced the peak AHP. The dual effect of zero K+ was reflected by the biphasic time course of voltage responses to zero K+: an inhibitory phase with enhanced peak AHP and slower firing, followed by a delayed excitatory phase with faster rate of membrane depolarization and faster firing. In the presence of strophanthidin to block the sodium pump, zero K+ consistently decreased firing by enhancing the peak AHP. Repetitive applications of K+ -free solution gradually turned the biphasic inhibitory-followed-by-excitatory voltage response into a monophasic inhibitory response in cells recorded with the whole cell (but not the cell-attached) mode, suggesting rundown of sodium pump activity. Taken together, the results suggest that spontaneous firing of SCN neurons is regulated by sodium pump activity as well as the AHP, and that sodium pump activity is modulated by intracellular soluble substances subject to rundown under the whole cell conditions.

Diurnal modulation of the Na+/K+-ATPase and spontaneous firing in the rat retinorecipient clock neurons.

The ventral "core" suprachiasmatic nucleus (vSCN) neurons are the retinorecipient neurons in the mammalian circadian clock and maintain a diurnal firing rhythm in reduced preparations. We tested the possibility that daily changes in Na+/K+-ATPase accompany diurnal variation in spontaneous electrical activity. In control, bath application of 9 microM strophanthidin increased the spontaneous firing both at day and night but to different extents. In the presence of 1 mM Ni2+ to block spontaneous firing, addition of 9 microM strophanthidin, but not higher concentrations (6.5-20 mM) of external K+, induced the silenced cells to fire action potentials in a diurnal rhythm, suggesting a diurnal change in Na+/K+-ATPase activity. Consistently, voltage-clamp recordings demonstrated that the pump current blocked by 9 microM strophanthidin was approximately three times larger in daytime than nighttime and was little affected by the presence of 1 mM Ni2+. Experiments with various concentrations of strophanthidin further suggests day-night differences in maximum Na+/K+-ATPase activity, amounting to 6 pA of pump current at day and down to 3.5 pA at night, and in its half-block concentrations, changing from a daytime value of 4 microM to a nighttime value of 8 microM. Our results indicate that the vSCN neurons exhibit a diurnal rhythm in the Na+/K+-ATPase the activity of which is higher during the day when the firing rate is also higher. Mechanistically, the modulation could be accounted for in terms of changes in the maximum activity of Na+/K+-ATPase and its ability to block by strophanthidin.

A pathophysiologic study of the kidney tubule to optimize organ preservation solutions.

BACKGROUND: Tissue damage at the time of organ transplantation has a negative impact on the subsequent success of the procedure, both in the immediate and longer term. Hypothermia is the principal element used to prolong organ viability ex vivo, but paradoxically also induces cellular edema through inhibition of energy-dependent adenosine triphosphatases (ATPases). This induces an electrolyte imbalance that leads to fluid influx and cell swelling. It is important, therefore, that improvements are made in the preservation of ischemic organs to reduce this injury. METHODS: This study has applied a novel in vitro system to model cold and warm ischemic-induced renal tubule swelling that characterizes tissue damage in ischemia/reperfusion injury. Biochemical blockade of ATPases in this system using strophanthidin modeled the effects of energy depletion and induced cell swelling. By measuring such tubule swelling and changes to tubular cell volume in isolated rabbit renal proximal tubules, an analysis was made that defined the basis on which an optimal preservation solution may be developed. RESULTS: The data show that our model could reproduce ischemically induced cell swelling and characterized the response at the cellular level of tubules to different components of preservation solutions. The data indicate that an isosmolar, phosphate-buffered, sucrose solution prevented tubule swelling more effectively than Euro-Collins, hyperosmolar citrate, or University of Wisconsin solutions that are in routine clinical use. CONCLUSION: Future developments in organ preservation may significantly improve transplant outcomes. Our novel analysis forms the basis of future whole-organ studies that ultimately may allow us to propose an optimum platform for improved preservation solutions.