Electrogenic Sodium Pump Research Articles

Effect OF m-trifluoromethyl- attachment on the glutathione peroxidase mimicry and antioxidant actions of diphenyl diselenide: Essential thiols of electrogenic sodium pump as a mechanistic component

m-Ditrifluoromethyl-diphenyl diselenide [DFDD (m-CF3-C6H4Se)2] is a disubstituted diaryl analog of diphenyl diselenide [DPDS (C6H5Se)2] in which a hydrogen atom on each aromatic ring is replaced by trifluoromethyl group (-CF3). Herein, we investigated the effect of the -CF3 group introduction on the GPx mimetic and antioxidant properties of DPDS. Animals were euthanized, brains were removed, and used for lipid peroxidation, cerebral sodium pump activity and thiols assays in vitro. Results showed that DFDD utilizes exogenous thiols [dithiol treitol (DTT), cysteine (Cys) and glutathione (GSH)] to reduce hydroperoxides. Furthermore, DFDD only protected against deoxyribose degradation in the presence of DTT. DFDD also exerted marked (p< 0.05) inhibitory effect on Fe2+or H2O2 or fenton reaction-induced lipid peroxidation in rat cerebral tissue homogenate. In addition, DFDD simultaneously (p< 0.05) inhibited pump activity and lipid peroxidation in cerebral tissue homogenate assaulted with prooxidants with proportionate depletion of thiol in the reaction system. This assay was repeated in the presence of DTT or Cys-or GSH and results revealed that enzyme's activity was not inhibited indicating that DFDD switched from enzymes's thiols to the oxidation of medium's thiols. It is rational to conclude that the introduction of -CF3 group to the aromatic rings of DFDD does not abolish its GPx mimetic and antioxidant properties and these still rely on thiols of cerebral electrogenic sodium pump. DFDD could be a suitable candidate for relative pharmacological effect and weak toxicity consequent to its possession of high electron withdrawing group. However, further research is needed in this regard.

Approximate analytical solution of a (V,m,h) reduced system for backpropagating action potentials in sparsely excitable dendrites

We derive an approximate analytical solution of a nonlinear cable equation describing the backpropagation of action potentials in sparsely excitable dendrites with clusters of transiently activating, TTX-sensitive Na+ channels of low density, discretely distributed as point sources of transmembrane current along a continuous (non-segmented) passive cable structure. Each cluster or hot-spot, corresponding to a mesoscopic level description of Na+ ion channels, included known cumulative inactivation kinetics observed at the microscopic level. In such a reduced third-order system, the ‘recovery’ variable is an electrogenic sodium-pump and/or a Na+-Ca2+ exchanger imbedded in the passive membrane, and a high leakage conductance stabilizes the system. A nonlinear cable equation was used to investigate back-propagation and repetitive activity of action potentials, exhibiting characteristics of the modified Hodgkin-Huxley kinetics (in the presence of suprathreshold input). In particular, a time-dependent analytical solution was obtained through a perturbation expansion of the non-dimensional membrane potential (Φ) for all voltage dependent terms including the voltage dependent Na+ activation μ) and state-dependent inactivation (η) gating variables and then solving the resulting system of integral equations. It was shown that back-propagating action potentials attenuate in amplitude with the frequency following experimental findings and that the discrete and low-density distributions of transient Na+ channels along the cable structure contribute significantly to their discharge patterns. A major significance of integrative modelling is the provision of a continuous description of the non-dimensional membrane potential (Φ) as a function of position.

Nerve excitability differences in slow and fast motor axons of the rat: more than just Ih.

The objective was to determine biophysical differences between fast and slow motor axons using threshold tracking and demonstrate confounds related to anesthetic. Nerve excitability of motor axons innervating the slow-twitch soleus (SOL) and fast-twitch tibialis anterior (TA) muscles was tested. The experiments were conducted with pentobarbital sodium (SP) anesthetic and compared with previous results that used ketamine-xylazine (KX). Nerve excitability indices measured with SP show definitive differences between TA and SOL motor axons that extend beyond previous reports. Nerve excitability indices sensitive to changes in Ih indicated an increase in SOL axons compared with TA axons [e.g., S3 t = 7.949 (df = 10), P < 0.001; hyperpolarizing threshold electrotonus (90-100 ms), t = 2.659 (df = 20); P = 0.01; hyperpolarizing I/V slope, t = 4.308 (df = 19); P < 0.001]. SOL axons also had a longer strength-duration time constant [t = 3.35 (df = 20); P = 0.003] and a longer and larger magnitude relative refractory period [RRP (ms) t = 3.53 (df = 12); P = 0.004; Refractoriness at 2 ms, t = 0.0055 (df = 9); P = 0.006]. Anesthetic choice affected many measures of peripheral nerve excitability with differences most apparent in tests of threshold electrotonus and recovery cycle. For example, recovery cycle with KX lacked a clear superexcitable and late subexcitable period. We conclude that KX had a confounding effect on nerve excitability results consistent with ischemic depolarization. Results using SP revealed the full extent of differences in nerve excitability measures between putative slow and fast motor axons of the rat. These results provide empirical evidence, beyond conduction velocity, that the biophysical properties of motor axons vary with the type of muscle fiber innervated. These differences suggest that fast axons may be predisposed to dysfunction during hyperpolarizing stresses, e.g., electrogenic sodium pumping following sustained impulse conduction.NEW & NOTEWORTHY Nerve excitability testing is a tool used to provide insight into the properties of ion channels in peripheral nerves. It is used clinically to assess pathophysiology of axons. Researchers customarily think of motor axons as homogeneous; however, we demonstrate there are clear differences between fast and slow axons in the rat. This is important for interpreting results with selective motor neuronopathy, like aging where fast axons are at high risk of degeneration.

S118 Mechanisms of diabetic neuropathy

Objectives Diabetic polyneuropathy (DPN) is the most common peripheral neuropathy representing a severe disabling condition. There is currently no disease modifying treatment, and development of targeted therapies depends on understanding the complex underlying mechanisms of DPN. The objective of this review is to discuss mechanisms of DPN. Methods DPN is associated with disturbances in endoneurial metabolism and microvascular morphology, but the roles of these as the cause of DPN have been controversial. The scientific community has been divided into two groups, favoring either metabolic or neurovascular mechanisms. Results Since DPN is decreased by improved metabolic control, hyperglycemia is assumed to be the main cause. Long lasting hyperglycemia leads to a downstream metabolic cascade which affects mitochondrial function and causes an excess formation of reactive oxygen species and exaggerated oxidative stress. Additionally, dyslipidemia, insulin resistance, autoimmunity, and epigenetics are suggested to contribute to metabolic mechanisms of DPN. The idea that microvascular changes cause DPN was challenged in early studies. There is, however, increasing evidence that changes in endoneurial capillary morphology and vascular reactivity may predate the development of DPN. In recent studies, disturbances in capillary function were shown to reduce the amount of oxygen and glucose that can be extracted by the tissue for a given blood flow. Discussion The ability to show immediately the effects on patient axons of interventions such as ischemia makes excitability testing a powerful method to investigate disease pathophysiology. Significance Axonal excitability tests may contribute to understanding the mechanisms of diabetic neuropathy with the possibility to provide information about the activity of different ion channels and the electrogenic sodium pump.

S119 Axonal excitability studies in diabetic neuropathy

Diabetic polyneuropathy (DPN) is a complication of diabetes involving complex mechanisms. The introduction of automated threshold-tracking has led to a number of abnormal axonal excitability findings associated with diabetes without neuropathy (DWN) and DPN. The first abnormality described was a striking resistance to ischaemia in DWN. Superexcitability measurements showed that this ischaemic resistance was not due to a depolarized resting potential, but was related to the mean blood glucose over 24 h, indicating a rapid effect of glucose on nerve metabolism. Glycaemic control by insulin treatment in patients with diabetic neuropathy restored sensitivity to ischaemia. In contrast to DWN, patients with established DPN exhibited ‘fanning-in’ of threshold electrotonus and reduced superexcitability, indicating membrane depolarization. Using the rate of recovery of excitability following maximum voluntary contraction as a measure of electrogenic sodium pump activity, excitability measurements indicated that sodium pump function was normal in DWN, but reduced in DPN, accounting for the membrane depolarization. Further excitability studies have attempted to determine changes, other than resistance to ischaemia, that precede the development of neuropathy. Evidence that depolarization precedes neuropathy has been found both in Type 1 and Type 2 diabetics. An ischaemic cause was suggested by the finding that excitability abnormalities correlated better with vascular-related factors than with HgbA1c. Recently excitability studies have moved on from assessing pathophysiology to treatment. In Type 1 DWN, glycaemic control by continuous insulin infusion preserved membrane potential much better than comparable control by multiple injections, perhaps because glucose variability can cause microvascular dysfunction.

Inorganic mercury interacts with thiols at the nucleotide and cationic binding sites of the ouabain-sensitive cerebral electrogenic sodium pump

The molecular events leading to neuronal dysfunction often associated with mercury toxicity can be complex and is yet to be fully elucidated. Hence, the present study sought to evaluate the interaction of inorganic mercury (Hg 2+) with the ouabain-sensitive electrogenic pump in partially purified mammalian brain membrane preparations. The results show that Hg 2+ significantly inhibited the transmembrane enzyme in a concentration dependent manner. In addition, Hg 2+ exerts its inhibitory effect on the activity of the enzyme by interacting with groups at the adenosine triphosphate (ATP), Na + and K + binding sites. However, preincubation of the enzyme with exogenous monothiols, cysteine, prevented the inhibition of Hg 2+ on the pump's activity suggesting that Hg 2+ may be interacting with the thiols at the nucleotide (ATP) and cationic (Na + and K +) binding sites. In fact, our data show that Hg 2+ oxidizes sulphydryl groups in cysteine in a time dependent fashion in vitro. Finally, we speculate that the small molecular volume of Hg 2+ in comparison with the substrates (ATP, Na + and K +) of sodium pump, its possibly high reactivity and strong affinity for thiols may account for its high toxicity towards the membrane bound ouabain-sensitive electrogenic pump.

Growth of calcium-blind mutants of Yersinia pestis at 37 degrees C in permissive Ca2+-deficient environments.

Cells of wild-type Yersinia pestis exhibit a low-calcium response (LCR) defined as bacteriostasis with expression of a pCD-encoded type III secretion system (T3SS) during cultivation at 37 degrees C without added Ca(2+) versus vegetative growth with downregulation of the T3SS with Ca(2+) (>or=2.5 mM). Bacteriostasis is known to reflect cumulative toxicity of Na(+), l-glutamic acid and culture pH; control of these variables enables full-scale growth ('rescue') in the absence of Ca(2+). Several T3SS regulatory proteins modulate the LCR, because their absence promotes a Ca(2+)-blind phenotype in which growth at 37 degrees C ceases and the T3SS is constitutive even with added Ca(2+). This study analysed the connection between the LCR and Ca(2+) by determining the response of selected Ca(2+)-blind mutants grown in Ca(2+)-deficient rescue media containing Na(+) plus l-glutamate (pH 5.5), where the T3SS is not expressed, l-glutamate alone (pH 6.5), where l-aspartate is fully catabolized, and Na(+) alone (pH 9.0), where the electrogenic sodium pump NADH : ubiquinone oxidoreductase becomes activated. All three conditions supported essentially full-scale Ca(2+)-independent growth at 37 degrees C of wild-type Y. pestis as well as lcrG and yopN mutants (possessing a complete but dysregulated T3SS), indicating that bacteriostasis reflects a Na(+)-dependent lesion in bioenergetics. In contrast, mutants lacking the negative regulator YopD or the YopD chaperone (LcrH) failed to grow in any rescue medium and are therefore truly temperature-sensitive. The Ca(2+)-blind yopD phenotype was fully suppressed in a Ca(2+)-independent background lacking the injectisome-associated inner-membrane component YscV but not peripheral YscK, suggesting that the core translocon energizes YopD.

Beneficial versus harmful effects of Angiotensin (1-7) on impulse propagation and cardiac arrhythmias in the failing heart

The presence of Angiotensin (1-7) (Ang 1-7) and ACE 2 in the ventricle of cardiomyopathic hamsters as well as the influence of Ang (1-7) on membrane potential, impulse propagation and cardiac excitability were investigated. Histology and immunochemistry were used to demonstrate the presence of Ang (1-7) and ACE 2 in the ventricle of cardiomyopathic hamsters. Measurements of transmembrane potentials, conduction velocity and refractoriness were made using conventional intracellular microelectrodes. The influence of Ang (1-7) on sodium pump current was investigated in voltageclamped myocytes isolated from the ventricle. The results indicated the presence of Ang (1-7) and ACE 2 in myocytes of cardiomyopathic hamsters. Moreover,Ang (1-7) (10(-8) M) hyperpolarised the heart cell, increased the conduction velocity, and reduced transiently the action potential duration. The cardiac refractoriness was also increased by the heptapeptide, an effect in part reduced by an inhibitor of mas receptor. These findings indicate that Ang (1-7) has important antiarrhythmic properties. However, the beneficial effects of Ang (1-7) are dose-dependent because at higher concentration (10(-7) M) the heptapeptide elicited an appreciable increase of action potential duration and early-after depolarisations. Since losartan (10(-7) M) did not counteract this effect of the high dose of the heptapeptide, it is possible to conclude that activation of AT(1)-receptors is not involved in this effect of Ang (1-7). To investigate the mechanism of the hyperpolarising action of Ang (1-7) the influence of the heptapeptide on the sodium potassium pump current was studied in myocytes isolated from the ventricle of cardiomyopathic hamsters. The peak pump current density was measured under voltage clamp using the whole cell configuration. The results indicated that Ang (1-7) (10(-8) M) enhanced the electrogenic sodium pump, an effect suppressed by ouabain (10(-7) M). Ang (1-7) has beneficial effects on the failing heart by activating the sodium pump, hyperpolarising the cell membrane and increasing the conduction velocity. These effects as well as the increment of refractoriness indicate that Ang (1-7) has antiarrhythmic properties. At higher concentrations (10(-7) M), however, the heptapeptide induced early-after depolarisations which leads to the conclusion that an optimal generation of Ang (1-7) must be achieved to permit a protective role of Ang (1-7) on cardiac arrhythmias.

Beneficial Effect of Eplerenone on Cardiac Remodelling and Electrical Properties of the Failing Heart

The effect of chronic administration of eplerenone on cardiac remodelling and electrical properties was investigated in the failing heart of cardiomyopathic hamsters (TO-2) at five months of age. Two-month-old hamsters were treated with eplerenone (200 mg/kg/day) administered into the chow for a period of three months. Measurements of membrane potential were performed with intracellular microelectrodes connected to a high impedance DC amplifier. The thickness of the ventricular wall as well as the area of fibrosis were measured. To investigate the influence of eplerenone on the electrogenic sodium pump myocytes were isolated from the ventricle and the pump current density was measured in voltage clamped cells using the whole cell clamp configuration. The results indicated that: 1) the width of the left and right ventricular wall was significantly reduced; 2) the heart weight/body weight ratio was decreased by 38+/-2.4% (n=24) (p<0.05); 3) the fibrotic area in the left ventricle (LV) was reduced by 12.6+/-2% (n=25) (p<0.05); 4) the incidence of cardiac arrhythmias was decreased from 58+/-3.8% (n=20) in the control to 40+/-4.1% (n=20) (p<0.05) in animals treated with eplerenone. Moreover, a significant reduction in the dispersion of the QT interval was found with the drug; 5) eplerenone increased the resting potential of ventricular fibres from 64.3+/-1.5 mV to 73.4+/-1.4 mV (n=30) (p<0.05), an effect related to the activation of an electrogenic sodium pump. The conduction velocity, in longitudinal direction, was enhanced from 50+/-2.2 cm/s (n=10) in the controls to 59+/-2.4 cm/s (n=13) (p<0.05) in animals treated with eplerenone. Eplerenone reduces cardiac remodelling, the incidence of cardiac arrhythmias and improves impulse propagation, an effect in part related to the antifibrotic effect of the drug but also to the activation of the electrogenic sodium pump.

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.

Angiotensin (1-7) re-establishes impulse conduction in cardiac muscle during ischaemia-reperfusion. The role of the sodium pump

The effect of angiotensin (1-7) (Ang 1-7) on membrane potential and excitability of rat heart muscle under ischaemia/reperfusion was investigated. The hearts of adult rats were removed under deep anaesthesia and perfused using the Langendorff method. After 40 minutes of global no-flow ischaemia, the heart was reperfused for five minutes and the right ventricle was dissected out and transferred to a transparent chamber, through which normal oxygenated Krebs solution flowed continuously (37 degrees C). Measurements of membrane potential were performed using an intracellular microelectrode connected to a high impedance preamplifier. The muscle was stimulated with rectangular current pulses (3 ms duration; 0.6 Hz) generated by an electronic stimulator and isolation unit. To study the influence of Ang (1-7) on sodium pump current, isolated myocytes were voltage-clamped at -40 mV and the current generated by the pump was recorded before and after the administration of Ang (1-7) (10-8 M) to the bath. Ang (1-7) (10-8 M) hyperpolarised the ischaemic heart fibre and re-established impulse propagation. The increment of resting potential was related to the activation of the sodium pump. Indeed, Ang (1-7) (10-8 M) enhanced the transient outward current generated by an electrogenic sodium pump. Both effects of Ang (1-7) on membrane potential and pump current were abolished by ouabain (10-7 M). The cardiac refractoriness was also increased by Ang (1-7) (10-8 M). Ang (1-7) activates the sodium pump, hyperpolarises the heart cell and re-establishes the impulse conduction during ischaemia/reperfusion. These effects of Ang (1-7), and the increment of cardiac refractoriness, provide an explanation for the reduced incidence of arrhythmias during ischaemia/reperfusion in the presence of Ang (1-7).

Voltage dependence of the apparent affinity for external Na(+) of the backward-running sodium pump.

The steady-state voltage and [Na(+)](o) dependence of the electrogenic sodium pump was investigated in voltage-clamped internally dialyzed giant axons of the squid, Loligo pealei, under conditions that promote the backward-running mode (K(+)-free seawater; ATP- and Na(+)-free internal solution containing ADP and orthophosphate). The ratio of pump-mediated (42)K(+) efflux to reverse pump current, I(pump) (both defined by sensitivity to dihydrodigitoxigenin, H(2)DTG), scaled by Faraday's constant, was -1.5 +/- 0.4 (n = 5; expected ratio for 2 K(+)/3 Na(+) stoichiometry is -2.0). Steady-state reverse pump current-voltage (I(pump)-V) relationships were obtained either from the shifts in holding current after repeated exposures of an axon clamped at various V(m) to H(2)DTG or from the difference between membrane I-V relationships obtained by imposing V(m) staircases in the presence or absence of H(2)DTG. With the second method, we also investigated the influence of [Na(+)](o) (up to 800 mM, for which hypertonic solutions were used) on the steady-state reverse I(pump)-V relationship. The reverse I(pump)-V relationship is sigmoid, I(pump) saturating at large negative V(m), and each doubling of [Na(+)](o) causes a fixed (29 mV) rightward parallel shift along the voltage axis of this Boltzmann partition function (apparent valence z = 0.80). These characteristics mirror those of steady-state (22)Na(+) efflux during electroneutral Na(+)/Na(+) exchange, and follow without additional postulates from the same simple high field access channel model (Gadsby, D.C., R.F. Rakowski, and P. De Weer, 1993. Science. 260:100-103). This model predicts valence z = nlambda, where n (1.33 +/- 0.05) is the Hill coefficient of Na binding, and lambda (0.61 +/- 0.03) is the fraction of the membrane electric field traversed by Na ions reaching their binding site. More elaborate alternative models can accommodate all the steady-state features of the reverse pumping and electroneutral Na(+)/Na(+) exchange modes only with additional assumptions that render them less likely.

Quantification of the α 3 subunit of the Na +/K +-ATPase in developing rat cerebellum

Cerebellar Purkinje neurons of rats have been shown to exhibit a progressive increase in resting membrane potential as the animals develop postnatally. The magnitude of this increase was equivalent in magnitude to the increase in the depolarizing action of ouabain, consistent with a role for the Na +/K +-pump in the hyperpolarization. Ouabain binding sites in whole cerebellum also increased with age. The present study was undertaken to confirm that the increases in ouabain binding and the electrophysiological responses to ouabain were a consequence of increases in the sodium pump and to determine whether the changes seen at the whole organ level were reflective of changes taking place at the cellular level. Using antibodies directed against the α 1, α 2, and α 3 subunits of the Na +/K +-ATPase, rats between 13 and 19 days of age exhibited a statistically significant increase in the relative amount of the α 3 subunit at the level of the whole organ, as determined by Western and slot blot analyses, with no change in the levels of either the α 1 or the α 2 subunit . Using immunohistochemistry, the α 3 subunit was shown to increase in both the Purkinje cell layer and the white matter during this postnatal time period, while the α 1 subunit increased in the granular layer. These results support and extend previous work, which pointed to a role for the electrogenic sodium pump in the developmental increase in Purkinje cell membrane potential. Furthermore, the data provide a cellular mechanism underlying the increase in resting membrane potential, that is, by the specific modulation of the α 3 subunit isoform.

The electrogenic sodium pump activity in Aplysia neurons is not potential dependent

We have investigated the potential dependence of the electrogenic sodium pump in Aplysia neurons by recording the potential and current induced by sudden change of the artificial sea water from one containing K+ at various concentrations to K+ -free sea water in the presence or absence of ouabain. Both K+ free sea water and ouabain block sodium transport and result in a significant depolarization due to removal of a maintained outward current that is a result of transport of more Na+ out of the cell than K+ into the cell during pump operation. In the presence of ouabain there is, however, an inward current induced by changing external K+ concentration from zero to some value between 1 and 20 mM, and this current is greater with a greater K+ concentration gradient. The current induced by change from zero to 1 mM K+ does not show any potential dependence, although those currents induced by higher K+ concentrations are potential dependent. We conclude that the activity of the electrogenic sodium pump is not potential dependent, but that the potential independence is obscured if higher concentrations of K+ are used to activate the electrogenic sodium pump.

The role of the sodium pump in the developmental regulation of membrane electrical properties of cerebellar Purkinje neurons of the rat

The postnatal development of the rat cerebellum has been well described morphologically and functionally. However, information regarding the electrical characteristics of Purkinje neurons during development is sparse. Using standard intracellular recording, the basic electrical properties of Purkinje neurons in cerebellar slices were compared at different postnatal ages. There was a significant, progressive increase in the resting membrane potential (RMP) of Purkinje neurons with age as well as a small but significant decrease in the cellular input resistance (Rin). The cardiac glycoside, ouabain (1 mM), an inhibitor of the sodium pump, depolarized Purkinje neurons significantly more with age. The magnitude of the increase in depolarizing activity of ouabain was equivalent to the magnitude of the increase in membrane potential. The number of ouabain binding sites was also found to increase with age suggesting an age related increase in the number of sodium pump sites. These results suggest that the predominant cellular mechanism which underlies the increase in membrane potential of Purkinje neurons during development is an increase in the density of Na +, K + pump sites and in the contribution of the electrogenic sodium pump.

A method for measuring resting and action potentials with external electrodes in crayfish giant axons

The true resting membrane potential and membrane action potential of crayfish giant axons were recorded with two external electrodes using a partition method in which the insulation grade was extensively improved by utilizing a water-absorbing particle mixed with vaseline and liquid paraffin, and the residual membrane potential at the lateral reference pool was removed by using an offset solution. The offset solution contained 10-4M of ouabain to block electrogenic sodium pump and a higher potassium concentration than that of the isotonic KCl solution. No short circuiting correction was necessary within an error of a few percent. Elimination of chloride ions from the offset solution substantially prolonged the surviving time of the preparation. This experimental system was developed to study a cell in as intact condition as possible, especially in a long-term experiment.

Purification and Reconstitution of Na+-translocating Vacuolar ATPase from Enterococcus hirae

Vacuolar ATPases make up a family of proton pumps distributed widely from bacteria to higher organisms. An unusual member of this family, a sodium-translocating ATPase, has been found in the eubacterium Enterococcus hirae. We report here the purification of enterococcal Na+-ATPase from the plasma membrane of cells, whose ATPase content was highly amplified by expression of the cloned ntp operon that encodes this Na+-ATPase (ntpFIKECGABDHJ). The purified enzyme appears to consist of nine Ntp polypeptides, all the above except for the ntpH and ntpJ gene products. ATPase activity was strictly dependent on the presence of Na+ or Li+ ions and was inhibited by nitrate, N-ethylmaleimide, and the peptide antibiotic destruxin B. When the purified ATPase was reconstituted into liposomes prepared from Enterococcus faecalis phospholipids, ATP-driven Na+ uptake was observed; uptake was blocked by nitrate, destruxin B, and monensin, but it accelerated by carbonyl cyanide m-chlorophenylhydrazone and valinomycin. These data demonstrate that E. hirae Na+-ATPase is an electrogenic sodium pump of the vacuolar type. This is a promising system for research on the fundamental molecular structure and mechanism of vacuolar ATPase.

Pharmacological block of the electrogenic sodium pump disrupts rhythmic bursting induced by strychnine and bicuculline in the neonatal rat spinal cord.

The cellular mechanisms underlying rhythmic bursts induced in the isolated neonatal rat spinal cord by bath application of strychnine and bicuculline (which block glycine- and gamma-aminobutyric acid-A-receptor-mediated inhibition, respectively) were probed with pharmacological tools. Such spontaneous bursts were recorded either intracellularly from lumbar motoneurons or extracellularly from ventral roots. As previously described, these network-driven events consisted of large-amplitude depolarizations arising abruptly from baseline with a highly regular period (on average 28 s). Burst episodes (lasting on average 7 s) comprised several oscillations and appeared synchronously on flexor and extensor motoneuron pools of both sides of the spinal cord. Their diffuse location made convenient to use bath-applied substances in the attempt to selectively block distinct membrane processes operating through the network. Application of apamin (0.4 microM) shortened both cycle period and burst duration without changing their regular rhythmicity. Similar results were obtained with carbachol (10 microM). Cs+ (4 mM) reversibly hyperpolarized the motoneuron membrane potential and largely increased burst duration, which was characterized by a long series of repetitive oscillatory waves. Cycle period and rhythmicity remained unaltered. Ouabain (10 microM), strophanthidin (4 microM), or K(+)-free solutions disrupted rhythmic bursting, which was fragmented into irregularly occurring paroxysmal activity mixed with short depolarizing events, still developing simultaneously on both sides of the spinal cord. Bursting activity eventually ceased after approximately 30-40 min of application of ouabain or strophanthidin. Prolonged washout of strophanthidin or K(+)-free solutions reestablished regular bursting patterns, whereas no recovery from ouabain was observed. At the time of strong depression of bursting, it was still possible to evoke bursts by single electrical pulses applied to the segmental dorsal root. Antidromic spikes of motoneurons could still be evoked by ventral root stimulation. These results demonstrate that, in a spinal bursting network mainly made up by excitatory processes, blockers of slow Ca(2+)-dependent K+ currents, such as apamin or carbachol, or of the slow inward rectifier, such as Cs+, did not suppress rhythmicity, suggesting that these conductances simply contributed to control cycle period and/or burst duration. Conversely, pharmacological blockers of the electrogenic Na+ pump such as ouabain, strophanthidin, or K(+)-free solutions severely disrupted all characteristics of rhythmic bursting. It is proposed that the operation of the electrogenic Na+ pump of premotoneurons was a crucial element for rhythmic bursting.

Bursting firing of action potentials in central snail neurons elicited by d-amphetamine: role of the electrogenic sodium pump

The effects of d-amphetamine on central neurons were studied electrophysiologically in the identifiable RP4 neuron of the African snail, Achatina fulica Ferussac. d-Amphetamine elicited bursting activity from the central RP4 neuron in a concentration-dependent manner. The bursting activity was not blocked in a high magnesium (30 mM) medium, or after a continuous perfusion of propranolol, prazosin, haloperidol, phenobarbital, hexamethonium, d-tubocurarine, atropine, or calcium-free solution containing EDTA or verapamil. These results suggested that the bursting activity elicited by d-amphetamine was not due to: (1) the synaptic effects of neurotransmitters; or (2) the cholinergic or adrenergic receptors of the excitable membrane. However, the bursting activity elicited by d-amphetamine was blocked in the presence of ouabain or in the medium containing potassium-free, low sodium solutions, d-Amphetamine did not elicit the bursting activity of the LP4 neuron in the same ganglia preparation, and did not alter the GABA-elicited currents of the snail neuron. It is concluded, therefore, that d-amphetamine induced a potassium- and sodium-dependent bursting activity of central neurons. The bursting activity of the central neuron may be associated with the sodium pump of the neuron.

Electrogenic Na + transport by Enterococcus hirae Na +-ATPase

Energy-dependent generation of a membrane potential (Δψ) (−45 mV, interior negative) was observed in the F 0F 1, H +-ATPase-defective mutant of Enterococcus hirae. The generation of Δψ was found at high pH (but not at low pH), for which intracellular Na + was required but not extracellular K +. The Δψ-generating activity was induced in cells cultured in media containing high concentrations of Na +, and was not observed in the Na +-ATPase mutants. These results suggest that E. hirae Na +-ATPase is responsible for the electrogenic sodium pump.