Electrolyte Levels in Neoplastic and Nonneoplastic Human Urothelium After Ouabain Treatment: X-Ray Microanalysis of Bulk Hydrated Samples

Samples of neoplastic and nonneoplastic human urothelium were immediately frozen, incubated in Krebs' saline and then frozen, or incubated in 10^?5 mol/L ouabain in Krebs' saline and then frozen. The frozen specimens were then planed in a cryoultramicrotome and examined by low-temperature scanning electron microscopy. X-Ray microanalysis was performed on the superficial urothelial cells. Neoplastic cells immediately frozen and those incubated in Krebs' saline had significantly higher K⁺/Na⁺, K⁺/P, and K⁺/Cl^? ratios and lower Na⁺/P and Cl^?/P ratios than nonneoplastic cells. Incubation in ouabain led to a fall in the K⁺/Na⁺, K⁺/P, and K⁺/Cl^? ratios and a rise in the Na⁺/P and Na⁺/Cl^? ratios in both neoplastic and nonneoplastic cells and effectively nullified the difference between them. These results are consistent with the concept that in neoplasia a primary event is stimulation of Na⁺/H⁺ exchange, which leads to secondary stimulation of the ouabain-sensitive Na⁺/K⁺ ATPase pump.     

The Na/K-ATPase as [K]o sensor: Role in cardiovascular disease pathogenesis and augmented production of endogenous cardiotonic steroids

Current evidence demonstrates that augmented production of endogenous cardiotonic steroids (CTS) such as ouabain and marinobufagenin is involved in the pathogenesis of hypertension and other cardiovascular diseases associated with volume expansion. It is also well documented that the development of hypertension and the cardiovascular complications of this disease are provoked by hypokalemia and suppressed by high-K⁺ diet. We hypothesized that altered extracellular K⁺ (K⁺)_o handling contributes to the pathogenesis of hypertension via modulation of interaction of endogenous CTS with Na⁺/K⁺-ATPase. To examine this hypothesis, experiments were performed with C7-Madin-Darby canine kidney epithelial cells at [K⁺]_o detected in plasma under control conditions (4.5 mM), severe hypokalemia (2 mM), and hyperkalemia (7 mM). Elevation of [K⁺]_o from 2 to 7 mM increased the threshold of modulation of intracellular (Na⁺)_i and (K⁺)_i content by ouabain from 1 to 10 nM, which corresponds to the range of endogenous CTS detected in plasma from patients with volume-expanded disorders. In control medium, 30% activation of cell proliferation was observed with 3 nM ouabain, whereas the addition of 0.3 nM ouabain was sufficient to induce about the same increment of cell proliferation in K⁺-depleted medium. [K⁺]_o elevation up to 7 mM completely abolished the proliferative effect of ouabain. At [K⁺]_o = 2, 4.5 and 7 mM, the death of ouabain-treated cells was indicated in the presence of 10, 30 and 300 nM ouabain, respectively. In conclusion, our results showed that modulation of [K⁺]_o in a pathophysiologically reasonable range sharply affected efficacy of endogenous CTS in the elevation of the [Na⁺]_i/[K⁺]_i ratio and in triggering (Na⁺)_i,(K⁺)_i-independent signaling resulting in cell proliferation and death. We propose that Na⁺/K⁺-ATPase may be considered as a [K⁺]_o sensor involved in the crosstalk of (K⁺)_o handling with the pathogenesis of cardiovascular diseases.     

Preliminary characterization of an Na,K,Cl co-transport activity in cultured human astrocytes

We have studied the potassium uptake using ⁸⁶Rb⁺ into monolayers of secondary cultures of human astrocytes prepared from cerebral hemispheres of a 4-month-old fetus. With the use of inhibitors we could attribute 30–40% of the ⁸⁶Rb⁺ uptake to an Na⁺,K⁺-ATPase, 50–60% to an anion-cation co-transporter and 10% to potassium leak channels. The anion-cation co-transporter was dependent on the simultaneous presence of both sodium and chloride in the incubation medium and is therefore most likely an Na⁺,K⁺,Cl⁻ co-transporter. This is the first evidence of such an Na⁺,K⁺,Cl⁻ co-transport in human astrocytes.     

Pharmacological dissection of receptor-associated and voltage-sensitive ionic channels involved in catecholamine release

The experiments were designed to quantify pharmacologically the degree of participation of channels associated with the nicotinic cholinoceptor compared with voltage-sensitive channels during the evoked release of [³H]noradrenaline from prelabelled 3–7-day old cultured bovine adrenal chromaffin cells. To achieve this purpose we studied (a) the release of [³H]noradrenaline evoked by secretagogues known to trigger the secretory response through activation of receptor-associated channels (acetylcholine, nicotine), voltage-sensitive Na⁺ (veratridine) and Ca²⁺ (high [K⁺]) channels or direct, channel-independent promotion of Ca²⁺ entry (ionomycin); and (b) the selective blockade of some of those responses using ionic manipulations (Na⁺ deprivation, high Mg²⁺) or drugs known to block the activity of receptoroperated channels (imipramine, cocaine), voltage-dependent Na⁺ (tetrodotoxin) or Ca²⁺ (nitrendipine) channels.

Inhibition by nitrendipine, a potent Ca²⁺ antagonist, of the secretory responses to both nicotine and high [K⁺] indicates a preferential Ca²⁺ entry through voltage-sensitive channels during the secretory process. Blockade by cocaine and imipramine of the release of [³H]noradrenaline evoked by acetylcholine and nicotine, without alteration of the responses to high [K⁺], veratridine or ionomycin, speaks in favor of a selective inactivation of the nicotinic receptor-associated channel. Since Na⁺ deprivation abolished [³H]noradrenaline release produced by nicotine, it seems that Na⁺ entry through the receptor-linked ionophore might be a primary event in the initiation of the secretory process; the fact that tetrodotoxin did not affect the release favors this view.

However, veratridine induced a tetrodotoxin-sensitive secretory response, suggesting the presence of voltage-sensitive Na⁺ channels which might physiologically be used to propagate action potentials through gap junctions between adjacent chromaffin cells, only in the intact gland. Propagated action potentials will activate voltage-sensitive Ca²⁺ channels, producing a massive Ca²⁺ entry and an explosive ejection of catecholamines simultaneously from many cells; during stressful situations this could constitute an efficient mechanism for the amplification of the secretory signal initiated by depolarization of chromaffin cells at restricted subsynaptic areas by acetylcholine liberated at splanchnic nerve-chromaffin cell junctions.     

线粒体酶活性及线粒体肿胀与不同运动强度的关系

背景：关于不同运动强度对机体的影响早有研究,并且取得了不少有价值的成果。但是对于不同运动强度对线粒体Na⁺,K⁺-ATP酶、Ca2⁺-ATP酶活性以及线粒体肿胀程度影响的研究较少。 目的：观察不同运动强度对大鼠骨骼肌线粒体Na⁺,K⁺-ATP酶和Ca2⁺-ATP酶以及线粒体肿胀的影响。 方法：将24只SD大鼠随机分为正常对照组,中等强度运动组和高强度运动组。正常对照组大鼠正常笼内生活,不运动。另2组游泳训练方式,分别建立中强度和高强度运动模型,进行相应的运动锻炼,训练4周。 结果与结论：中强度运动组线粒体Na⁺,K⁺-ATP酶、Ca2⁺-ATP酶活性与正常对照组相比均增高(P < 0.05),线粒体肿胀程度降低(P < 0.05);而高强度运动组线粒体Na⁺,K⁺-ATP酶、Ca2⁺-ATP酶活性与正常对照组相比均降低(P < 0.05),线粒体肿胀程度增大(P < 0.05)。实验结果表明中强度运动可以保护线粒体Na⁺,K⁺-ATP酶、Ca2⁺-ATP酶的活性,提高线粒体功能;而高强度运动则降低了线粒体的功能。     

Comparison of the effects of a bufodienolide and ouabain on neuronal and smooth muscle preparations

The effect of a bufodienolide (monohydroxy-14,15-epoxy-20,22-dienolide glycoside) purified from toad skin was compared with that of ouabain on ³H-noradrenaline release and on the tension of rabbit pulmonary arterial strips. This compound exerted an ouabain-like activity. The neuronal effects of this bufodienolide derivative on squid axon were also studied and compared with those of ouabain. Both compounds enhanced the resting and stimulation-evoked (2 Hz. 360 shocks) release of ³H-noradrenaline. Moreover, in the presence of either this bufodienolide or ouabain, the tension of the rabbit artery increased gradually, and the contraction evoked by electrical stimulation was potentiated. Both compounds enhanced, in a prazosin-sensitive way, smooth muscle responses to noradrenaline and to electrical stimulation. In higher concentrations, they contracted smooth muscle cells of pulmonary artery, an action which was insensitive to prazosin. The bufodienolide was about 8 times more active in inhibition of ²²Na efflux than was ouabain, but did not affect Ca efflux, which is not sensitive to ouabain. It is therefore concluded that compounds with an inhibitory effect on Na⁺,K⁺-ATPase are able to affect chemical neurotransmission of blood vessels in such a way that in lower concentrations they potentiate the release of noradrenaline, and in higher concentrations they contract directly the smooth muscle. These findings indicate that such compounds if they are present in the circulation might be involved in the physiological regulation of blood pressure or in the genesis of hypertension.     

Bretylium causes a K(+)-Na+ pump activation that is independent of Na+/H+ exchange in depolarized rat, mouse and human lymphocytes

We have studied a bretylium tosylate induced increase of the membrane potentials of partially depolarized rat, mouse and human lymphocytes, using the potential sensitive dye, bis [1,3, dibutylbarbituric acid-(5) trimethine oxonol]. The extent of this repolarization is dose-dependent and decreased in magnitude as the temp was reduced from 37°C to room temp. The repolarizing effect is inhibited by K⁺-Na⁺-pump blockers or lack of extracellular Na⁺.

Sodium ion channel blockers are effective in abolishing repolarization only if applied prior to, or simultaneously with, bretylium. Activation of Na⁺/H⁺ exchange is not involved in the mechanism of the phenomenon as the latter is completely eliminated in the presence of 10μM amiloride (concn of the diuretics having no measurable inhibition on the action of the exchanger). These data suggest that bretylium opens ligand- and voltage-gated Na⁺ channels, and repolarization occurs due to higher activity of the K⁺-Na⁺-pump stimulated by the enhanced intracellular Na⁺ accumulation.     

Ion channels and the control of swimming in the Xenopus embryo

The Xenopus embryo has been well studied and the circuitry underlying motor pattern generation largely elucidated. We have extended this analysis by determining the roles of individual voltage- and ligand-gated ion channels in controlling the motor pattern for swimming and two mechanisms that control rundown of this pattern.

Xenopus embryo spinal neurons possess at least six classes of ion channel: a fast Na⁺ channel; a mixture of kinetically similar Ca²⁺ channels; a fast K⁺ channels; a slow K⁺ channel; a Na⁺-dependent K⁺ channel; and a slowly activating Ca²⁺-dependent K⁺ channel.

The roles of the voltage-gated currents in determining neuronal firing properties and operation of the locomotor circuitry have been examined both pharmacologically and in realistic computer simulations.

Model neurons fire repetitively in response to current injection. The Ca²⁺ current seems essential for repetitive firing. The fast K⁺ current appears mainly to control spike width, whereas the slow K⁺ current exerts a powerful influence on repetitive firing. These predictions from the model have been confirmed by the use of specific pharmacological blockers of the fast and slow K⁺ currents.

Both the model network and the real spinal locomotor circuit appear to tolerate a wide variation in the relative strengths of the component synapses but are very sensitive to the magnitudes of the voltage-gated currents. In particular the slow K⁺ current, despite being a small component of the total outward current, plays a critical role in stabilizing the motor pattern.

Like many other rhythmic motor patterns, swimming in the Xenopus embryo is episodic; it undergoes run-down and self-termination even in the absence of sensory inputs. The slow Ca²⁺-dependent K⁺ current appears to play a role in the self-termination of swimming. However, intrinscic modulation mediated by the release of ATP and production of adenosine in the extracellular space appears to be a very powerful determinant of run-down of the motor pattern.     

Disruption of ionic and cell volume homeostasis in cerebral ischemia: The perfect storm

The mechanisms of brain tissue damage in stroke are strongly linked to the phenomenon of excitotoxicity, which is defined as damage or death of neural cells due to excessive activation of receptors for the excitatory neurotransmitters glutamate and aspartate. Under physiological conditions, ionotropic glutamate receptors mediate the processes of excitatory neurotransmission and synaptic plasticity. In ischemia, sustained pathological release of glutamate from neurons and glial cells causes prolonged activation of these receptors, resulting in massive depolarization and cytoplasmic Ca²⁺ overload. High cytoplasmic levels of Ca²⁺ activate many degradative processes that, depending on the metabolic status, cause immediate or delayed death of neural cells. This traditional view has been expanded by a number of observations that implicate Cl⁻ channels and several types of non-channel transporter proteins, such as the Na⁺,K⁺,2Cl⁻ cotransporter, Na⁺/H⁺ exchanger, and Na⁺/Ca²⁺ exchanger, in the development of glutamate toxicity. Some of these ion transporters increase tissue damage by promoting pathological cell swelling and necrotic cell death, while others contribute to a long-term accumulation of cytoplasmic Ca²⁺. This brief review is aimed at illustrating how the dysregulation of various ion transport processes combine in a ‘perfect storm’ that disrupts neural ionic homeostasis and culminates in the irreversible damage and death of neural cells. The clinical relevance of individual transporters as targets for therapeutic intervention in stroke is also briefly discussed.     

The neurotoxicity of ouabain, a sodium-potassium ATPase inhibitor, in the rat hippocampus

Intrahippocampal injection of 1 nmol ouabain, a sodium/potassium (Na⁺,K⁺-)ATPase inhibitor, produced a necrotic lesion within 4 days, characterised by a massive invasion by foaming macrophages. A lower dose of ouabain (0.1 nmol) produced a more discrete lesion of all groups of neuronal perikarya in the hippocampus, with only a minimal degree of glial infiltration. The neuronal perikaryal death produced in the subicular, CA1 and CA2 regions was only partially decreased by intraperitoneal injections of the anticonvulsants diazepam and MK-801; these drugs were without effect in the CA3 or hilar interneuronal regions. At neither dose of ouabain was there any indication of neuronal loss in brain regions outside the hippocampus, typically produced by prolonged seizure activity. It is suggested that ouabain has a two-fold action, a release of toxic acidic amino acids and a prolonged depolarization of neurons leading to osmolysis or calcium necrosis.     

Mechanism of histamine-induced calcium efflux from cultured bovine adrenal chromaffin cells: possible involvement of an Na/Ca exchange mechanism

The effect of stimulation of the histamine receptor on Ca²⁺ mobilization in cultured bovine adrenal chromaffin cells was examined. Histamine (10⁻⁵ M) increased the intracellular free Ca²⁺ ([Ca²⁺]_i) to a peak in the presence or absence of extracellular Ca²⁺, followed by decrease with time. Histamine (10⁻⁸–10⁻⁵ M) also stimulated ⁴⁵Ca²⁺ efflux from cultured bovine adrenal chromaffin cells in a concentration dependent manner. Its stimulatory effect on ⁴⁵Ca²⁺ efflux was inhibited by the specific histamine H₁ receptor antagonist mepyramine. The increase in histamine-stimulated ⁴⁵Ca²⁺ efflux was inhibited by deprivation of extracellular Na⁺ and by the Na⁺/Ca²⁺ exchange inhibitor amiloride. In addition, histamine stimulated ²²Na⁺ influx into the cells, and this action was inhibited by amiloride. These results suggest that stimulation of the histamine H₁ receptor regulates Na⁺/Ca²⁺ exchange in cultured bovine adrenal chromaffin cells.     

窦房结膜钟和钙钟在缺血条件下对细胞自律性的调控

急性心肌缺血导致的窦性心动过缓甚至停搏在临床上较为常见,但对其微观机制尚缺乏足够的认识。在窦房结细胞动作电位动力学数学模型的基础上,通过提高胞外K⁺和胞内Na⁺浓度,增强L 型Ca²⁺电流I_CaL,降低Na Ca交换电流I_NCX以及T 型Ca²⁺电流I_CaT模拟缺血,定量研究各因素对起搏细胞自律性的作用。另外,基于单域方程构建一维窦房结及心房组织模型,研究窦房结缺血时组织中的电传导状态以及钙钟的作用。结果表明,缺血时I_CaL的增强以及胞外K⁺浓度的蓄积对自律性并无明显的影响。I_CaT降低1倍后尽管可使起搏频率降低13%,而自律性仍可维持。但是,当细胞内Na^+升高10%,或I_NCX减小55%后,会出现电位的振荡乃至停搏。进一步的研究说明,胞内Na⁺的蓄积间接地通过降低I_NCX对自律性产生调控作用。因此, I_NCX的降低应是缺血时造成窦性心动过缓及停搏的主要原因。研究表明,在缺血条件下,增强钙钟的活动有助于自律性和电传导的恢复,表明膜钟和钙钟的相互作用仍是构成细胞自动除极的重要基础。     

Aging: Stimulation rate on cardiac intracellular Na activity and developed tension

Previous reports suggested that Na,K-ATPase activity and Na⁺-pump capacity decrease with senescence in left atrial myocardium of F344 rats. Current experiments were designed to determine if this reduction in the Na⁺-pump affects free intracellular Na⁺ levels. Mean intracellular Na⁺ ion activity (aⁱ_Na) was measured with Na-selective microelectrodes in left atrial muscle isolated from hearts of 4-, 14- and 25-month-old F344 rats. Preparations were stimulated randomly at frequencies between 0 and 12 h. There were no age-associated differences in a ⁱ_Na measured at any frequency or in the decay of Na⁺ activity following discontinuation of electrical stimulation. These data indicate that the aging-related decline in Na,K-ATPase does not result in elevated aⁱ_Na even at extremely high stimulation frequencies, thus suggesting that other routes of Na⁺ influx and efflux are also altered in atrial muscle.     

Characterization of the norovirus 3C-like protease

The recombinant 3C-like protease of Chiba virus, a Norovirus, expressed in Escherichia coli cells was purified and characterized as to effects of pH, temperature, salt contents, and SH reagents on its proteolytic activity. The optimal pH and temperature of the 3C-like protease for the proteolytic activity were 8.6 and 37 °C, respectively. Increased concentration (100 mM) of monovalent cations such as Na⁺ and K⁺ was inhibitory to the activity. Hg²⁺ and Zn²⁺ remarkably inhibited the protease activity, while Mg²⁺ and Ca²⁺ had no virtual effect. Several sulfhydryl reagents such as p-chloromercuribenzoic acid, methyl methanethiosulfonate, N-ethylmaleimide and N-phenylmaleimide also blocked the activity, confirming the previous result that cysteine residue(s) were responsible for the proteolysis.     

In vivo release of somatostatin from rat median eminence after local K infusion or delivery of nociceptive stress

The effects of local infusion of a 16 mM K⁺ solution or of a nociceptive stress on the release of somatostatin (SRIF) from the hypothalamus was measured in unanesthetized male rats implanted with a push-pull cannula in the median eminence. Although the baseline secretion rate of SRIF was increased in animals displaying agitation as a result of handling stress, both treatments induced fast doubling of SRIF release lasting for 15–30 min. Neither an equimolar Na⁺ infusion into the median eminence nor a similar K⁺ infusion into the 3rd ventricle had any affect on this release. The possible role of SRIF release in the mechanism of growth hormone inhibition following nociceptive stress is discussed.     

A proton pumping pyrophosphatase in acidocalcisomes of Herpetomonas sp

Acidocalcisomes are acidic calcium storage organelles found in several microorganisms. They are characterized by their acidic nature, high electron density, high content of polyphosphates and several cations. Electron microscopy contrast tuned images of Herpetomonas sp. showed the presence of several electron dense organelles ranging from 100 to 300 nm in size. In addition, X-ray element mapping associated with energy-filtering transmission electron microscopy showed that most of the cations, namely Na, Mg, P, K, Fe and Zn, are located in their matrix. Using acridine orange as an indicator dye, a pyrophosphate-driven H⁺ uptake was measured in cells permeabilized by digitonin. This uptake has an optimal pH of 6.5–6.7 and was inhibited by sodium fluoride (NaF) and imidodiphosphate (IDP), two H⁺-pyrophosphatase inhibitors. H⁺ uptake was not promoted by ATP. Addition of 50 μM Ca²⁺ induced the release of H⁺, suggesting the presence of a Ca²⁺/H⁺ countertransport system in the membranes of the acidic compartments. Na⁺ was unable to release protons from the organelles. The pyrophosphate-dependent H⁺ uptake was dependent of ion K⁺ and inhibited by Na⁺ Herpetomonas sp. immunolabeled with monoclonal antibodies raised against a Trypanosoma cruzi V-H⁺-pyrophosphatase shows intense fluorescence in cytoplasmatic organelles of size and distribution similar to the electron-dense vacuoles.

Together, these results suggest that the electron dense organelles found in Herpetomonas sp. are homologous to the acidocalcisomes described in other trypanosomatids. They possess a vacuolar H⁺-pyrophosphatase and a Ca²⁺/H⁺ antiport. However, in contrast to the other trypanosomatids so far studied, we were not able to measure any ATP promoted H⁺ transport in the acidocalcisomes of this parasite.     

Role of K+ Ion Channels in Lymphokine-Activated Killer (LAK) Cell Lytic Function

Cells of the immune system possess K⁺ ion channels which have been implicated in various cellular functions including activation, differentiation and cytolytic function. To define the role of K⁺ ion channels in the lytic function of lymphokine-activated killer (LAK) cells, we investigated the effects of K⁺ channel blockers on their cytolytic activity. Results show that when LAK cell mediated cytolysis of AKIL-20 tumor cexis was carried out in the presence of: a) the K⁺ channel blocker, 4-aminopyridine (4-AP); b) the monoamine, serotonin (5-hydroxytryptamine; 5-HT); c) the serotonin agonist, quipazine; d) or the Ca⁺⁺ dependent K⁺ Channel blocker, quinidine, the cytolytic activity of the LAK cells was inhibited in a dose-dependent manner. Preincubation of LAK effector cells also inhibited lysis in a dose-dependent manner, whereas preincubation of the AKIL-20 tumor target cells produced no inhibitory effects. This study demonstrates that K⁺ ion channels are involved in the LAK cell cytolytic process and that compounds, including neuroendocrine products, which modulate K⁺ ion channel function are capable of modulating the lytic activity of these effector cells.     

The local anaesthetic QX-314 enables enhanced whole-cell recordings of excitatory synaptic currents in rat hippocampal slices in vitro

Whole-cell voltage-clamped currents were recorded using low resistance patch electrodes containing K⁺ channel blockers with the local anaesthetic QX-314 (20mM). Under these conditions voltage-gated Na⁺ conductances and regenerative spiking activity were blocked, leaving Ca²⁺ conductances. The effect of QX-314 resembled that of extracellular tetrodotoxin but had the advantage of being localised to one cell therefore permitting recording of synaptic currents. Excitatory synaptic current recordings were progressively improved over the first 25 minutes of recording as input resistance increased. After this time postsynaptic currents were stable for at least 1 h.     

Dual effect of verapamil on K-evoked release of endogenous dopamine from arcuate nucleus-median eminence complex

The effect of verapamil, a calcium-entrance blocker, on K⁺-evoked release of endogenous dopamine from tuberoinfundibular neurons incubated in vitro was studied. This compound, added to the incubation medium, at the dose of 10⁻⁶ M, significantly reinforced K⁺-induced dopamine release, whereas, at higher doses (10⁻⁵, 5 × 10⁻⁵ and 10⁻⁴ M), it completely prevented the stimulated dopamine release. The results obtained with the higher doses showed the calcium dependence of K⁺-evoked release of endogenous dopamine from central neurons. The opposite effect, seen with the lower dose of verapamil, could be due to different pharmacological properties of the drug.     

Alteration of acylphosphatase levels in familial Alzheimer''s disease fibroblasts with presenilin gene mutations

Acylphosphatase (ATPase), an enzyme that modulates the activity of Ca²⁺-ATPase by hydrolysing its phosphorylated moiety, has been found to be significantly higher in cultured skin fibroblasts from donors affected by early onset familial Alzheimer's disease (EOFAD) with PS-1 and PS-2 gene mutations. Of the two known isoenzymes of acylphosphatase, only the erythrocyte one accounts for the total increase in activity. No relevant alteration was observed in phosphotyrosine phosphatase activity (PTPase), in Ca²⁺-ATPase and Na⁺,K⁺-ATPase activities of the same cells as compared to age-matched controls. This finding could suggest a possible explanation for the calcium-dependent biochemical alterations previously described in Alzheimer's disease fibroblasts.