The role of extracellular potassium dynamics in the different stages of ictal bursting and spreading depression: A computational study

Experimental evidences point out the participation of nonsynaptic mechanisms (e.g., fluctuations in extracellular ions) in epileptiform bursting and spreading depression (SD). During these abnormal oscillatory patterns, it is observed an increase of extracellular potassium concentration [K⁺]_o and a decrease of extracellular calcium concentration [Ca²⁺]_o which raises the neuronal excitability. However, whether the high [K⁺]_o triggers and propagates these abnormal neuronal activities or plays a secondary role into this process is unclear. To better understand the influence of extracellular potassium dynamics in these oscillatory patterns, the experimental conditions of high [K⁺]_o and zero [Ca²⁺]_o were replicated in an extended Golomb model where we added important regulatory mechanisms of ion concentration as Na⁺-K⁺ pump, ion diffusion and glial buffering. Within these conditions, simulations of the cell model exhibit seizure-like discharges (ictal bursting). The SD was elicited by the interruption of the Na⁺−K⁺ pump activity, mimicking the effect of cellular hypoxia (an experimental protocol to elicit SD, the hypoxia-induced SD). We used the bifurcation theory and the fast-slow method to analyze the interference of K⁺ dynamics in the cellular excitability. This analysis indicates that the system loses its stability at a high [K⁺]_o, transiting to an elevated state of neuronal excitability. Effects of high [K⁺]_o are observed in different stages of ictal bursting and SD. In the initial stage, the increase of [K⁺]_o creates favorable conditions to trigger both oscillatory patterns. During the neuronal activity, a continuous growth of [K⁺]_o by outward K⁺ flow depresses K⁺ currents in a positive feedback way. At the last stage, due to the depression of K⁺ currents, the Na⁺-K⁺ pump is the main mechanism in the end of neuronal activity. Thus, this work suggests that [K⁺]_o dynamics may play a fundamental role in these abnormal oscillatory patterns.     

The enhancement by pervanadate of tyrosine phosphorylation on prostatic proteins occurs through the inhibition of membrane-associated tyrosine phosphatases

The Na⁺–K⁺ ATPase activity and SH group content were decreased whereas malondialdehyde (MDA) content was increased upon treating the porcine cardiac sarcolemma with xanthine plus xanthine oxidase, which is known to generate superoxide and other oxyradicals. Superoxide dismutase either alone or in combination with catalase and mannitol fully prevented changes in SH group content but the xanthine plus xanthine oxidase-induced depression in Na⁺–K⁺ ATPase activity as well as increase in MDA content were prevented partially. The Lineweaver-Burk plot analysis of the data for Na⁺–K⁺ ATPase activity in the presence of different concentrations of MgATP or Na⁺ revealed that the xanthine plus xanthine oxidase-induced depression in the enzyme activity was associated with a decrease in V_max and an increase in K_m for MgATP; however, K_a value for Na⁺ was decreased. Treatment of sarcolemma with H₂O₂ plus Fe²⁺, an hydroxyl and other radical generating system, increased MDA content but decreased both Na⁺–K⁺ ATPase activity and SH group content; mannitol alone or in combination with catalase prevented changes in SH group content fully but the depression in Na⁺–K⁺ ATPase activity and increase in MDA content were prevented partially. The depression in the enzyme activity by H₂O₂ plus Fe²⁺ was associated with a decrease in V_max and an increase in K_m for MgATP. These results indicate that the depressant effect of xanthine plus xanthine oxidase on sarcolemmal Na⁺–K⁺ ATPase may be due to the formation of superoxide, hydroxyl and other radicals. Furthermore, the oxyradical-induced depression in Na⁺–K⁺ ATPase activity may be due to a decrease in the affinity of substrate in the sarcolemmal membrane.     

Disruption of Ion-Trafficking System in the Cochlear Spiral Ligament Prior to Permanent Hearing Loss Induced by Exposure to Intense Noise: Possible Involvement of 4-Hydroxy-2-Nonenal as a Mediator of Oxidative Stress

Noise-induced hearing loss is at least in part due to disruption of endocochlear potential, which is maintained by various K⁺ transport apparatuses including Na⁺, K⁺-ATPase and gap junction-mediated intercellular communication in the lateral wall structures. In this study, we examined the changes in the ion-trafficking-related proteins in the spiral ligament fibrocytes (SLFs) following in vivo acoustic overstimulation or in vitro exposure of cultured SLFs to 4-hydroxy-2-nonenal, which is a mediator of oxidative stress. Connexin (Cx)26 and Cx30 were ubiquitously expressed throughout the spiral ligament, whereas Na⁺, K⁺-ATPase α1 was predominantly detected in the stria vascularis and spiral prominence (type 2 SLFs). One-hour exposure of mice to 8 kHz octave band noise at a 110 dB sound pressure level produced an immediate and prolonged decrease in the Cx26 expression level and in Na⁺, K⁺-ATPase activity, as well as a delayed decrease in Cx30 expression in the SLFs. The noise-induced hearing loss and decrease in the Cx26 protein level and Na⁺, K⁺-ATPase activity were abolished by a systemic treatment with a free radical-scavenging agent, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl, or with a nitric oxide synthase inhibitor, N^ω-nitro-L-arginine methyl ester hydrochloride. In vitro exposure of SLFs in primary culture to 4-hydroxy-2-nonenal produced a decrease in the protein levels of Cx26 and Na⁺, K⁺-ATPase α1, as well as Na⁺, K⁺-ATPase activity, and also resulted in dysfunction of the intercellular communication between the SLFs. Taken together, our data suggest that disruption of the ion-trafficking system in the cochlear SLFs is caused by the decrease in Cxs level and Na⁺, K⁺-ATPase activity, and at least in part involved in permanent hearing loss induced by intense noise. Oxidative stress-mediated products might contribute to the decrease in Cxs content and Na⁺, K⁺-ATPase activity in the cochlear lateral wall structures.     

Interleukin-1β inhibits voltage-gated sodium currents in a time- and dose-dependent manner in cortical neurons

Interleukin-1β (IL-1β) is a multifunctional proinflammatory cytokine that plays a key role in the injuries and diseases of the central nervous system (CNS). A voltage-gated Na⁺ channel is essential for the excitability and electrical properties of neurons. However, it is not known whether IL-1β directly affects the central Na⁺ channels. In the present study, we examined the effects of IL-1β on Na⁺ currents in cultured cortical neurons using patch-clamp recording. Our results showed that IL-1β suppressed Na⁺ currents through its receptor in a time- and dose-dependent manner, but did not alter the voltage-dependent activation and inactivation. PKC and then p38 MAPK were involved in this inhibition. The spike amplitude was also inhibited by IL-1β in the doses that decreased the Na⁺ currents. Our findings revealed the inhibition of chronic IL-1β treatment on voltage-gated Na⁺ channels in the CNS, and showed that the action potential (AP) amplitude was reduced by IL-1β due to a decrease of Na⁺ currents.     

Prostaglandin E2 modulates Na+,K+-ATPase activity in rat hippocampus: implications for neurological diseases

Prostaglandin E₂ (PGE₂) is quantitatively one of the major prostaglandins synthesized in mammalian brain, and there is evidence that it facilitates seizures and neuronal death. However, little is known about the molecular mechanisms involved in such excitatory effects. Na⁺,K⁺‐ATPase is a membrane protein which plays a key role in electrolyte homeostasis maintenance and, therefore, regulates neuronal excitability. In this study, we tested the hypothesis that PGE₂ decreases Na⁺,K⁺‐ATPase activity, in order to shed some light on the mechanisms underlying the excitatory action of PGE₂. Na⁺,K⁺‐ATPase activity was determined by assessing ouabain‐sensitive ATP hydrolysis. We found that incubation of adult rat hippocampal slices with PGE₂ (0.1–10 μM) for 30 min decreased Na⁺,K⁺‐ATPase activity in a concentration‐dependent manner. However, PGE₂ did not alter Na⁺,K⁺‐ATPase activity if added to hippocampal homogenates. The inhibitory effect of PGE₂ on Na⁺,K⁺‐ATPase activity was not related to a decrease in the total or plasma membrane immunocontent of the catalytic α subunit of Na⁺,K⁺‐ATPase. We found that the inhibitory effect of PGE₂ (1 μM) on Na⁺,K⁺‐ATPase activity was receptor‐mediated, as incubation with selective antagonists for EP1 (SC‐19220, 10 μM), EP3 (L‐826266, 1 μM) or EP4 (L‐161982, 1 μM) receptors prevented the PGE₂‐induced decrease of Na⁺,K⁺‐ATPase activity. On the other hand, incubation with the selective EP2 agonist (butaprost, 0.1–10 μM) increased enzyme activity per se in a concentration‐dependent manner, but did not prevent the inhibitory effect of PGE₂. Incubation with a protein kinase A (PKA) inhibitor (H‐89, 1 μM) and a protein kinase C (PKC) inhibitor (GF‐109203X, 300 nM) also prevented PGE₂‐induced decrease of Na⁺,K⁺‐ATPase activity. Accordingly, PGE₂ increased phosphorylation of Ser943 at the α subunit, a critical residue for regulation of enzyme activity. Importantly, we also found that PGE₂ decreases Na⁺,K⁺‐ATPase activity in vivo. The results presented here imply Na⁺,K⁺‐ATPase as a target for PGE₂‐mediated signaling, which may underlie PGE₂‐induced increase of brain excitability.     

Multipurpose Na+ ions mediate excitation and cellular homeostasis: Evolution of the concept of Na+ pumps and Na+/Ca2+ exchangers

Ionic signalling is the most ancient form of regulation of cellular functions in response to environmental challenges. Signals, mediated by Na⁺ fluxes and spatio-temporal fluctuations of Na⁺ concentration in cellular organelles and cellular compartments contribute to the most fundamental cellular processes such as membrane excitability and energy production. At the very core of ionic signalling lies the Na⁺-K⁺ ATP-driven pump (or NKA) which creates trans-plasmalemmal ion gradients that sustain ionic fluxes through ion channels and numerous Na⁺-dependent transporters that maintain cellular and tissue homeostasis. Here we present a brief account of the history of research into NKA, Na⁺ -dependent transporters and Na⁺ signalling.     

Tryptic digest of the α subunit of lamb kidney (Na+ + K+)-ATPase

The M_r ≈ 100 000 α subunit was prepared from highly purified lamb kidney (Na⁺+ K⁺)-ATPase. Its N-terminal sequence is Gly-Arg-Asx-Lys-Tyr-Glu. The α subunit was S-carboxymethylated, succinylated, and cleaved at its 40 arginine residues with trypsin. Four major, well-differentiated peptide fractions (A to D) were obtained by chromatography of the digest on a Sephadex G-50 column. Fraction A eluted at the void volume of the column and contained aggregated, very hydrophobic peptides, possibly from regions of α that are buried within the membrane lipid bilayer in the native enzyme. Fractions B to D, which together accounted for about 75% of the total protein, contained water-soluble peptides. To test the feasibility of using antibodies to identify and purify specific peptides of α subunit, studies were carried out using antibodies to native (Na⁺+ K⁺)-ATPase. Carboxymethylation and succinylation did not significantly decrease total antibody binding to α subunit, although the affinity of the anti-(Na⁺ + K⁺)-ATPase antibodies for α subunit was reduced by about 50%. The tryptic peptides of a subunit also retain significant immunochemical reactivity. Fractions A, B and C (but not D) of the digest all bind antibodies. To characterize further the tryptic digest, 16 peptides from fraction D were isolated and sequence studies on these were carried out.     

Net sodium fluxes change significantly at anatomically distinct root zones of rice (Oryza sativa L.) seedlings

Casparian bands of endodermis and exodermis play crucial roles in blocking apoplastic movement of ions and water into the stele of roots through the cortex. These apoplastic barriers differ considerably in structure and function along the developing root. The present study assessed net Na⁺ fluxes in anatomically distinct root zones of rice seedlings and analyzed parts of individual roots showing different Na⁺ uptake. The results indicated that anatomically distinct root zones contributed differently to the overall uptake of Na⁺. The average Na⁺ uptake in root zones in which Casparian bands of the endo- and exo-dermis were interrupted by initiating lateral root primordia (root zone III) was significantly greater than that at the root apex, where Casparian bands were not yet formed (root zone I), or in the region where endo- and exo-dermis with Casparian bands were well developed (root zone II). The measurement of net Na⁺ fluxes using a non-invasive scanning ion-selective electrode technique (SIET) demonstrated that net Na⁺ flux varied significantly in different positions along developing rice roots, and a net Na⁺ influx was obvious at the base of young lateral root primordia. Since sodium fluxes changed significantly along developing roots of rice seedlings, we suggest that the significantly distinct net Na⁺ flux profile may be attributed to different apoplastic permeability due to lateral root primordia development for non-selective apoplastic bypass of ions along the apoplast.     

The interactions of ouabain with post-tetanic and facilitatory drug potentiations at cat soleus neuromuscular junctions in vivo

Cat soleus motor nerve terminals, after high frequency conditioning, generate a post-tetanic repetition (PTR) which leads to a post-tetanic (PTP) of the muscle response. This property enables quantitative assessment of enhancement or depression of this nerve terminal excitability in vivo. The present study focuses on ionic mechanisms underlying the PTRs produced in this neuromuscular system either by high frequency stimulation or edrophonium. Ouabain was used as a specific probe for inhibition of Na⁺–K⁺ ATPase and its known consequences on Na⁺ and Ca²⁺ translocation. Ouabain pretreatment doubled the duration over which single stimuli, following either high frequency or edrophonium conditioning produced PTR. Ouabain in the doses used had no effectper se but as a function of dose augmented the frequency dependent responses. This pointed to Na⁺ loading of nerve terminals via high frequency stimulation plus ouabain inhibition of Na⁺–K⁺ ATPase. Ouabain potentiation of PTR responses evidently depends on exchange of intra-terminal sodium for external calcium. Thus, calcium entry blockers, Mn²⁺, and Co²⁺ suppressed or abolished the potentiations both before and after ouabain. Diphenylhydantoin, a Na⁺ and Ca²⁺ blocker, acted similarly. The effects of stimulation frequency, ouabain and the sequence of events leading to PTR in the soleus neuromuscular system appeared in general no different from those derived from the many in vitro microphysiologic studies of this phenomenon. Thus, EPPs were augmented and prolonged. It was concluded that intracellular Ca²⁺ is critical for regulating the stability of systems in which repetitive firing is both a normal and abnormal function.Special issue dedicated to Dr. Sidney Udenfriend     

Ischemia-Related Subcellular Redistribution of Sodium Channels Enhances the Proarrhythmic Effect of Class I Antiarrhythmic Drugs: A Simulation Study

Background

Cardiomyocytes located at the ischemic border zone of infarcted ventricle are accompanied by redistribution of gap junctions, which mediate electrical transmission between cardiomyocytes. This ischemic border zone provides an arrhythmogenic substrate. It was also shown that sodium (Na⁺) channels are redistributed within myocytes located in the ischemic border zone. However, the roles of the subcellular redistribution of Na⁺ channels in the arrhythmogenicity under ischemia remain unclear.

Methods

Computer simulations of excitation conduction were performed in a myofiber model incorporating both subcellular Na⁺ channel redistribution and the electric field mechanism, taking into account the intercellular cleft potentials.

Results

We found in the myofiber model that the subcellular redistribution of the Na⁺ channels under myocardial ischemia, decreasing in Na⁺ channel expression of the lateral cell membrane of each myocyte, decreased the tissue excitability, resulting in conduction slowing even without any ischemia-related electrophysiological change. The conventional model (i.e., without the electric field mechanism) did not reproduce the conduction slowing caused by the subcellular Na⁺ channel redistribution. Furthermore, Na⁺ channel blockade with the coexistence of a non-ischemic zone with an ischemic border zone expanded the vulnerable period for reentrant tachyarrhythmias compared to the model without the ischemic border zone. Na⁺ channel blockade tended to cause unidirectional conduction block at sites near the ischemic border zone. Thus, such a unidirectional conduction block induced by a premature stimulus at sites near the ischemic border zone is associated with the initiation of reentrant tachyarrhythmias.

Conclusions

Proarrhythmia of Na⁺ channel blockade in patients with old myocardial infarction might be partly attributable to the ischemia-related subcellular Na⁺ channel redistribution.     

Effect of platelet-activating factor (PAF) on sodium calcium exchange in cardiac sarcolemmal vesicles

Summary The effects of platelet-activating factor (PAF) on Na⁺-dependent calcium uptake in myocardial sarcolemmal vesicles were examined in order to clarify its mechanism of inotropic action on the heart. PAF (40 and 20 µM) significantly inhibited Na⁺-Ca²⁺ exchange by 61% and 37%, respectively. Both initial rate of exchange and maximal exchange were inhibited. The Km for the reaction was not altered but Vmax was lowered 55% by PAF. Lyso-PAF inhibited Na⁺-Ca²⁺ exchange to a similar degree as PAF. CV-3988, a specific PAF receptor antagonist, failed to diminish the inhibitory effect of PAF on Na⁺-Ca²⁺ exchange, suggesting that the effect of PAF on Na⁺-Ca ²⁺ exchange is not via a receptor mechanism. The passive permeability of sarcolemmal vesicles to Ca²⁺ was markedly elevated after PAF treatment. However, this effect could not account for the decrease in Na⁺-Ca²⁺ exchange. Interestingly, passive Ca²⁺ binding to cardiac sarcolemma was increased by 40 µM PAF. This study indicates that a depression of Na⁺-Ca²⁺ exchange probably does not play a role in the negative inotropic effect of PAF on the myocardium under physiological conditions. Its mechanism of action on Na⁺-Ca²⁺ exchange is discussed.     

Conduction of the cardiac impulse. 3. Characteristics of very slow conduction

The excitability of short segments (5–7 mm) of bundles of canine Purkinje fibers was depressed by exposure to 15–18 mM K⁺, to 15–18 mM K⁺ plus 5 x 10^-6 epinephrine or norepinephrine, to low K⁺, and to low Na⁺. The depressed segment was in the center chamber of a three-chamber bath; the ends of the bundle were exposed to normal Tyrode solution. Each method of depression resulted in slow and probably decremental conduction with an effective conduction velocity in the middle chamber of about 0.05 m/sec, or one-way block, or two-way block with summation of the graded responses in the depressed region. The action potential in the depressed segment (the slow response) differs from the normal action potential in its response to applied stimuli. A second active depolarization can be evoked by cathodal stimulation during much of the slow response. The response in the depressed segment is graded. The response of depressed fibers may depend on excitatory events similar to those responsible for the slow component of the cardiac action potential. It is suggested that the slow response can propagate, at least decrementally, in fibers in which the rapid, Na⁺-dependent upstroke is absent, and can cause reentrant excitation by so doing.     

The Neuroprotective Effect of Curcumin and Nigella sativa Oil Against Oxidative Stress in the Pilocarpine Model of Epilepsy: A Comparison with Valproate

Oxidative stress has been implicated to play a role in epileptogenesis and pilocarpine-induced seizures. The present study aims to evaluate the antioxidant effects of curcumin, Nigella sativa oil (NSO) and valproate on the levels of malondialdehyde, nitric oxide, reduced glutathione and the activities of catalase, Na⁺, K⁺-ATPase and acetylcholinesterase in the hippocampus of pilocarpine-treated rats. The animal model of epilepsy was induced by pilocarpine and left for 22 days to establish the chronic phase of epilepsy. These animals were then treated with curcumin, NSO or valproate for 21 days. The data revealed evidence of oxidative stress in the hippocampus of pilocarpinized rats as indicated by the increased nitric oxide levels and the decreased glutathione levels and catalase activity. Moreover, a decrease in Na⁺, K⁺-ATPase activity and an increase in acetylcholinesterase activity occurred in the hippocampus after pilocarpine. Treatment with curcumin, NSO or valproate ameliorated most of the changes induced by pilocarpine and restored Na⁺, K⁺-ATPase activity in the hippocampus to control levels. This study reflects the promising anticonvulsant and potent antioxidant effects of curcumin and NSO in reducing oxidative stress, excitability and the induction of seizures in epileptic animals and improving some of the adverse effects of antiepileptic drugs.     

Effects of vasopressin and aldosterone on the lateral mobility of epithelial Na+ channels in A6 renal epithelial cells

We have previously demonstrated that apical Na⁺ channels in A6 renal epithelial cells are associated with spectrin-based membrane cytoskeleton proteins and that the lateral mobility of these channels, as determined by fluorescence photobleach recovery (FPR) analysis, is severely restricted by this association (Smith et al., 1991. Proc. Natl. Acad. Sci. USA 88:6971–6975). Recent data indicate that the actin component of the cytoskeleton may play a role in modulating Na⁺ channel activity (Cantiello et al., 1991. Am. J. Physiol. 261:C882–C888); however, it is unknown if the Na⁺ channel's linkage to the spectrin-based membrane cytoskeleton is also involved in regulating channel activity. In this study, we have used FPR to examine if the linkage of the Na⁺ channels to the membrane cytoskeleton is a site for modulation of Na⁺ channel activity in filter grown A6 cells by vasopressin and aldosterone. We hypothesized that if the linkage of the Na⁺ channels to the membrane cytoskeleton is a site for regulation of Na⁺ channel activity by vasopressin and aldosterone, then hormone-mediated changes in either the membrane cytoskeleton or the affinity of the Na⁺ channel for the membrane cytoskeleton, should be reflected in changes in the lateral mobility and/or mobile fraction of Na⁺ channels on the cell surface. FPR revealed that although the rates of lateral mobility were not affected, there was a twofold increase in mobility fraction (f) of apical Na⁺ channels in aldosterone-treated (16 hr) monolayers (f = 32.31 ± 5.42%) when compared to control (unstimulated) (f = 14.2 ± 0.77%) and vasopressin-treated (20 min) (f = 12.7 ± 2.4%) monolayers. The twofold increase in mobile fraction of Na⁺ channels corresponds to the average increase in Na⁺ transport in response to aldosterone in A6 cells. The aldosterone-induced increase in Na⁺ transport and mobile fraction can be inhibited by the methylation inhibitor, 3-deazaadenosine, consistent with the hypothesis that a methylation event is involved in aldosterone induced upregulation of Na⁺ transport. We propose that the membrane cytoskeleton is involved in the aldosterone-mediated activation of epithelial Na⁺ channels.Supported by NIH grants DK37206 (DJB), NS26733 and NS28072 (KJA), DK46705 (PRS) and AHA New York Affiliate grant 91007G (LCS).     

Ion Secretion and Isotonic Transport in Frog Skin Glands

The aim of this study was to clarify the mechanism of isotonic fluid transport in frog skin glands. Stationary ion secretion by the glands was studied by measuring unidirectional fluxes of ²⁴Na⁺, ⁴²K⁺, and carrier-free ¹³⁴Cs⁺ in paired frog skins bathed on both sides with Ringer's solution, and with 10⁻⁵ m noradrenaline on the inside and 10⁻⁴ m amiloride on the outside. At transepithelial thermodynamic equilibrium conditions, the ¹³⁴Cs⁺ flux ratio, J ^out _Cs/J ⁱⁿ _Cs, varied in seven pairs of preparations from 6 to 36. Since carrier-free ¹³⁴Cs⁺ entering the cells is irreversibly trapped in the cellular compartment (Ussing & Lind, 1996), the transepithelial net flux of ¹³⁴Cs⁺ indicates that a paracellular flow of water is dragging ¹³⁴Cs⁺ in the direction from the serosal- to outside solution. From the measured flux ratios it was calculated that the force driving the secretory flux of Cs⁺ varied from 30 to 61 mV among preparations. In the same experiments unidirectional Na⁺ fluxes were measured as well, and it was found that also Na⁺ was subjected to secretion. The ratio of unidirectional Na⁺ fluxes, however, was significantly smaller than would be predicted if the two ions were both flowing along the paracellular route dragged by the flow of water. This result indicates that Na⁺ and Cs⁺ do not take the same pathway through the glands. The flux ratio of unidirectional K⁺ fluxes indicated active secretion of K⁺. The time it takes for steady-state K⁺ fluxes to be established was significantly longer than that of the simultaneously measured Cs⁺ fluxes. These results allow the conclusion that — in addition to being transported between cells — K⁺ is submitted to active transport along a cellular pathway.Based on the recirculation theory, we propose a new model which accounts for stationary Na⁺, K⁺, Cl⁻ and water secretion under thermodynamic equilibrium conditions. The new features of the model, as compared to the classical Silva-model for the shark-rectal gland, are: (i) the sodium pumps in the activated gland transport Na⁺ into the lateral intercellular space only. (ii) A barrier at the level of the basement membrane prevents the major fraction of Na⁺ entering the lateral space from returning to the serosal bath. Thus, Na⁺ is secreted into the outside bath. It has to be assumed then that the Na⁺ permeability of the basement membrane barrier (P ^BM _Na) is smaller than the Na⁺ permeability of the junctional membrane (P ^JM _Na), i.e., P ^JM _Na/P ^BM _Na > 1. The secretory paracellular flow of water further requires that the Na⁺ reflection coefficients (σ_Na) of the two barriers are governed by the conditions, σ^BM _Na > 0, and σ^BM _Na > σ^JM _Na. (iii) Na⁺ channels are located in the apical membrane of the activated gland cells, so that a fraction of the Na⁺ outflux appearing downstream the lateral intercellular space is recirculated by the gland cells. Based on measured unidirectional fluxes, a set of equations is developed from which we estimate the ion fluxes flowing through major pathways during stationary secretion. It is shown that 80% of the sodium ions flowing downstream the lateral intercellular space is recycled by the gland cells. Our calculations also indicate that under the conditions prevailing in the present experiments 1.8 ATP molecule would be hydrolyzed for every Na⁺ secreted to the outside bath. Received: 30 January 1996/Revised: 12 March 1996     

Regulation of neuronal excitability by release of proteins from glial cells

Effects of glial cells on electrical isolation and shaping of synaptic transmission between neurons have been extensively studied. Here we present evidence that the release of proteins from astrocytes as well as microglia may regulate voltage-activated Na⁺ currents in neurons, thereby increasing excitability and speed of transmission in neurons kept at distance from each other by specialized glial cells. As a first example, we show that basic fibroblast growth factor and neurotrophin-3, which are released from astrocytes by exposure to thyroid hormone, influence each other to enhance Na⁺ current density in cultured hippocampal neurons. As a second example, we show that the presence of microglia in hippocampal cultures can upregulate Na⁺ current density. The effect can be boosted by lipopolysaccharides, bacterial membrane-derived stimulators of microglial activation. Comparable effects are induced by the exposure of neuron-enriched hippocampal cultures to tumour necrosis factor-α, which is released from stimulated microglia. Taken together, our findings suggest that release of proteins from various types of glial cells can alter neuronal excitability over a time course of several days. This explains changes in neuronal excitability occurring in states of thyroid hormone imbalance and possibly also in seizures triggered by infectious diseases.     

Roles of subcellular Na+ channel distributions in the mechanism of cardiac conduction

The gap junction and voltage-gated Na⁺ channel play an important role in the action potential propagation. The purpose of this study was to elucidate the roles of subcellular Na⁺ channel distribution in action potential propagation. To achieve this, we constructed the myocardial strand model, which can calculate the current via intercellular cleft (electric-field mechanism) together with gap-junctional current (gap-junctional mechanism). We conducted simulations of action potential propagation in a myofiber model where cardiomyocytes were electrically coupled with gap junctions alone or with both the gap junctions and the electric field mechanism. Then we found that the action potential propagation was greatly affected by the subcellular distribution of Na⁺ channels in the presence of the electric field mechanism. The presence of Na⁺ channels in the lateral membrane was important to ensure the stability of propagation under conditions of reduced gap-junctional coupling. In the poorly coupled tissue with sufficient Na⁺ channels in the lateral membrane, the slowing of action potential propagation resulted from the periodic and intermittent dysfunction of the electric field mechanism. The changes in the subcellular Na⁺ channel distribution might be in part responsible for the homeostatic excitation propagation in the diseased heart.     

Participation of protein phosphatases in regulation of sodium transport across the erythrocyte membrane of the lamprey Lampetra fluviatilis

Erythrocytes of lamprey Lampetra fluviatilis were incubated in standard isotonic medium at 20°C with ²²Na to determine the unidirectional Na⁺ influx. Cell incubation in the presence of various protein phosphatase inhibitors (NaF, cantharidin, calyculin A) led to a considerable increase of Na⁺ transport into erythrocytes. The stimulation of Na⁺ influx into erythrocytes rose with increase of concentration of calyculin A within the range of 10–100 nM. The calyculin A concentration producing a 50% activation of Na⁺ transport amounted to 41.5 nM. Under optimal experimental conditions, the Na⁺ influx increased from control level of 5–8 to 20–40 mmol/l cells/h under effect of protein phosphatase blockers. The Na⁺ transport induced by these inhibitors was completely suppressed on addition of amiloride to the incubation medium. The treatment of lamprey erythrocytes with protein phosphatase inhibitors was accompanied by a small (～12%), but statistically significant decrease of intracellular Na⁺ content. A small decrease of intracellular K⁺ content in erythrocyte was observed only under the effect of NaF. The obtained data allow making the conclusion that protein phosphatases of the PP1 and PP2A types play a significant role in regulation of Na⁺ transport across the lamprey erythrocyte membrane in both directions.     

Changes in fluid compartments and ionic composition in the isolated chick retina during SD

Total content of water, extracellular space (ES), Na⁺, K⁺, and Cl^– in the isolated chick retina were measured in the presence (test) or absence (control) of spreading depression (SD). During SD in medium with 0.5 mM or 2 mM MgSO₄, there is an increase in the intracellular concentration of Na⁺ and Cl^– and a decrease in the intracellular concentration of K⁺. A decrease in the ES was only found in the medium with 2 mM MgSO₄ together with a diminshed outmovement of K⁺. We suggest that a decrease in the ES is due to an increased absorption of K⁺ by the Muller cells, causing its swelling and consequently a decrease of the ES.The addition of sucrose (17 mM) to the incubation medium as the extracellular marker markedly decreased the intracellular concentration of Cl^– in control retinas, blocked the inward movement of this ion to the tissue during SD and also changed the K⁺ movement during the phenomenon in medium with 2 mM MgSO₄. We suggest that Cl^– is an important ion in the ionic balance of the Muller cells and that sucrose must have its site of action that these cells.     

Purification and characterization of an (Na++K+)-ATPase proteolipid labeled with a photoaffinity derivative of ouabain

Highly purified lamb kidney (Na⁺+K⁺)-ATPase was photoaffinity labeled with the tritiated 2-nitro-5-azidobenzoyl derivative of ouabain (NAB-ouabain). The labeled (Na⁺+K⁺)-ATPase was mixed with unlabeled carrier enzyme. Two proteolipid (γ1 and γ2) fractions were then isolated by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. The two fractions were interchangeable when rechromatographed on the LH-60 column, suggesting that γ1 is an aggregated form of γ2. The total yield was 0.8–1.5 mol of γ component per mol of catalytic subunit recovered. This indicates that the γ component is present in stoichiometric amounts in the (Na⁺+K⁺)-ATPase. The proteolipids that were labeled with NAB-ouabain copurified with the unlabeled proteolipids.