Modulation of Na(+),K(+) pumping and neurotransmitter uptake by beta-amyloid

Micromolar concentrations of beta-amyloid (Abeta), a 40/42-amino-acid-long proteolytic fragment (Abeta(1-40/42)) of the amyloid precursor protein, was shown previously to play a crucial role in pathogenesis of Alzheimer's disease. We used the Xenopus oocyte expression system to investigate specific effects of micromolar concentrations of Abeta(1-42) on the neurotransmitter transporters for gamma-aminobutyric acid (GABA), GAT1, and for the excitatory amino acid glutamate, EAAC1, which are driven by the transmembrane Na(+) gradient that is regulated by the Na(+),K(+)-ATPase. Brief treatment with Abeta(1-42), up to 80 min, leads to a significant inhibition of ion translocation by the Na(+),K(+)-ATPase (30-40%); also glutamate uptake is inhibited (20%) while GABA uptake is not affected. Since reduced glutamate uptake will result in elevated, neurotoxic concentrations of extracellular glutamate, we investigated the effects of Abeta(1-42) and the smaller fragments, Abeta(12-28) and Abeta(25-35), on EAAC1 in more detail. Prolonged incubation in 1 microM Abeta(1-42) leads to further, strong inhibition of glutamate uptake and EAAC1-mediated current (after 4 h inhibition amounts to more than 80%). Abeta(12-28) is less effective with 50% inhibition after 4 h of incubation at 20 microM. Abeta(1-42) and Abeta(12-28) affect EAAC1-mediated current to a similar extent as the rate of glutamate uptake. The effects on EAAC1-mediated current are irreversible if Abeta were applied for longer time periods. Peptides directly microinjected into the oocyte are ineffective suggesting that the observed effect were mediated by extracellular proteins. Abeta(25-35) hardly affects EAAC1-mediated current or glutamate uptake. The results demonstrate that Abeta specifically inhibits the Na(+),K(+) pump and EAAC1. The domain between amino acids 12 and 28 of Abeta seems to play a crucial role for inhibition of EAAC1. The inhibition of EAAC1 by neurotoxic, elevated extracellular glutamate levels may contribute to Alzheimer's pathogenesis.     

Na(+) and K(+) concentrations, extra- and intracellular voltages, and the effect of TTX in hypoxic rat hippocampal slices

Severe hypoxia causes rapid depolarization of CA1 neurons and glial cells that resembles spreading depression (SD). In brain slices in vitro, the SD-like depolarization and the associated irreversible loss of function can be postponed, but not prevented, by blockade of Na(+) currents by tetrodotoxin (TTX). To investigate the role of Na(+) flux, we made recordings from the CA1 region in hippocampal slices in the presence and absence of TTX. We measured membrane changes in single CA1 pyramidal neurons simultaneously with extracellular DC potential (V(o)) and either extracellular [K(+)] or [Na(+)]; alternatively, we simultaneously recorded [Na(+)](o), [K(+)](o), and V(o). Confirming previous reports, early during hypoxia, before SD onset, [K(+)](o) began to rise, whereas [Na(+)](o) still remained normal and V(o) showed a slight, gradual, negative shift; neurons first hyperpolarized and then began to gradually depolarize. The SD-like abrupt negative DeltaV(o) corresponded to a near complete depolarization of pyramidal neurons and an 89% decrease in input resistance. [K(+)](o) increased by 47 mM and [Na(+)](o) dropped by 91 mM. Changes in intracellular Na(+) and K(+) concentrations, estimated on the basis of the measured extracellular ion levels and the relative volume fractions of the neuronal, glial, and extracellular compartment, were much more moderate. Because [Na(+)](o) dropped more than [K(+)](o) increased, simple exchange of Na(+) for K(+) cannot account for these ionic changes. The apparent imbalance of charge could be made up by Cl(-) influx into neurons paralleling Na(+) flux and release of Mg(2+) from cells. The hypoxia-induced changes in interneurons resembled those observed in pyramidal neurons. Astrocytes responded with an initial slow depolarization as [K(+)](o) rose. It was followed by a rapid but incomplete depolarization as soon as SD occurred, which could be accounted for by the reduced ratio, [K(+)](i)/[K(+)](o). TTX (1 microM) markedly postponed SD, but the SD-related changes in [K(+)](o) and [Na(+)](o) were only reduced by 23 and 12%, respectively. In TTX-treated pyramidal neurons, the delayed SD-like depolarization took off from a more positive level, but the final depolarized intracellular potential and input resistance were not different from control. We conclude that TTX-sensitive channels mediate only a fraction of the Na(+) influx, and that some of the K(+) is released in exchange for Na(+). Even though TTX-sensitive Na(+) currents are not essential for the self-regenerative membrane changes during hypoxic SD, in control solutions their activation may trigger the transition from gradual to rapid depolarization of neurons, thereby synchronizing the SD-like event.     

Na(+) dependence and the role of glutamate receptors and Na(+) channels in ion fluxes during hypoxia of rat hippocampal slices

Spreading depression (SD) as well as hypoxia-induced SD-like depolarization in forebrain gray matter are characterized by near complete depolarization of neurons. The biophysical mechanism of the depolarization is not known. Earlier we reported that simultaneous pharmacological blockade of all known major Na(+) and Ca(2+) channels prevents hypoxic SD. We now recorded extracellular voltage, Na(+), and K(+) concentrations and the intracellular potential of individual CA1 pyramidal neurons during hypoxia of rat hippocampal tissue slices after substituting Na(+) in the bath by an impermeant cation, or in the presence of channel blocking drugs applied individually and in combination. Reducing extracellular Na(+) concentration [Na(+)](o) to 90 mM postponed the hypoxia-induced extracellular DC-potential deflection (DeltaV(o)) and reduced its amplitude, and it also postponed the SD-like depolarization of neurons. After lowering [Na(+)](o) to 25 mM, SD-like DeltaV(o) became very small, indicating that an influx of Na(+) is required for SD; influx of Ca(2+) ions alone is not sufficient. We then asked whether the SD-related Na(+) current flows through glutamate-controlled and/or through voltage-gated Na(+) channels. Administration of either the non-N-methyl-D-aspartate (NMDA) receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX), or the NMDA receptor antagonist (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) postponed the hypoxic DeltaV(o) and depressed its amplitude but the effect of the combined administration of these two drugs was not greater than that of either alone. During the early phase of hypoxia, before SD onset, [K(+)](o) increased faster and reached a much higher level in the presence of glutamate antagonists than in their absence. The [K(+)](o) level reached at the height of hypoxic SD was, however, not affected. When TTX was added to DNQX and CPP, SD was prevented in half the trials. When SD did occur, it was greatly delayed, yet eventually neurons depolarized to the same extent as in normal solution. The SD-related sudden drop in [Na(+)](o) was depressed by only 19% in the presence of the three drugs, indicating that Na(+) can flow into cells through pathways other than ionotropic glutamate receptors and TTX-sensitive Na(+) channels. We conclude that, when they are functional, glutamate-receptor-mediated and voltage-gated Na(+) currents are the major generators of the self-regenerative rapid depolarization, but in their absence other pathways can sometimes take their place. The final level of SD-like depolarization is determined by positive feedback and not by the number of channels available. A schematic flow chart of the events generating hypoxic SD is discussed.     

Role of oxidative stress in ischemia-reperfusion-induced changes in Na+,K(+)-ATPase isoform expression in rat heart

The aim of this study was to assess whether depression of cardiac Na+,K(+)-ATPase activity during ischemia/reperfusion (I/R) is associated with alterations in Na+,K(+)-ATPase isoforms, and if oxidative stress participates in these I/R-induced changes. Na+,K(+)-ATPase alpha1, alpha2, alpha3, beta1, beta2, and beta3 isoform contents were measured in isolated rat hearts subjected to I/R (30 min of global ischemia followed by 60 min of reperfusion) in the presence or absence of superoxide dismutase plus catalase (SOD+CAT). Effects of oxidative stress on Na+,K(+)-ATPase isoforms were also examined by perfusing the hearts for 20 min with 300 microM hydrogen peroxide or 2 mM xanthine plus 0.03 U/ml xanthine oxidase (XXO). I/R significantly reduced the protein levels of all alpha and beta isoforms. Treatment of I/R hearts with SOD+CAT preserved the levels of alpha2, alpha3, beta1, beta2, and beta3 isoforms, but not that of the alpha1 isoform. Perfusion of hearts with hydrogen peroxide and XXO depressed all Na+,K(+)-ATPase alpha and beta isoforms, except for alpha1. These results indicate that the I/R-induced decrease in Na+,K(+)-ATPase may be due to changes in Na+,K(+)-ATPase isoform expression and that oxidative stress plays a role in this alteration. Antioxidant treatment attenuated the I/R-induced changes in expression of all isoforms except alpha1, which appears to be more resistant to oxidative stress.     

Effect of cis-diaminedichloroplatinum (II) (cis-DDP) on the intracellular Na+/K(+)-ratio in K 562 leukemia cells as revealed by X-ray microanalysis.

Changes of intracellular ionic homeostasis are believed to play a role in the cytostatic action of cis-DDP. It has been observed by means of X-ray microanalysis that cis-DDP did not alter the intracellular Na+/K(+)-ratio of K 562 leukemia cells during incubation periods which lasted shorter than the average doubling time of the cells of nearly 15 h. After 24 h the treated cells displayed at least two main populations in the distribution histogram of the Na+/K(+)-ratio. The results indicated that the passage of cis-DDP through the plasma membrane by itself did not change the monovalent electrolyte balance at the early stage of its action in K 562 cells.     

Genetic modifications of seizure susceptibility and expression by altered excitability in Drosophila Na(+) and K(+) channel mutants

A seizure-paralysis repertoire characteristic of Drosophila "bang-sensitive" mutants can be evoked electroconvulsively in tethered flies, in which behavioral episodes are associated with synchronized spike discharges in different body parts. Flight muscle DLMs (dorsal longitudinal muscles) display a stereotypic sequence of initial and delayed bouts of discharges (ID and DD), interposed with giant fiber (GF) pathway failure and followed by a refractory period. We examined how seizure susceptibility and discharge patterns are modified in various K(+) and Na(+) channel mutants. Decreased numbers of Na(+) channels in nap(ts) flies drastically reduced susceptibility to seizure induction, eliminated ID, and depressed DD spike generation. Mutations of different K(+) channels led to differential modifications of the various components in the repertoire. Altered transient K(+) currents in Sh(133) and Hk mutants promoted ID induction. However, only Sh(133) but not Hk mutations increased DD seizure and GF pathway failure durations. Surprisingly, modifications in sustained K(+) currents in eag and Shab mutants increased thresholds for DD induction and GF pathway failure. Nevertheless, both eag and Shab, like Sh(133), increased DD spike generation and recovery time from GF pathway failure. Interactions between channel mutations with the bang-sensitive mutation bss demonstrated the role of membrane excitability in stress-induced seizure-paralysis behavior. Seizure induction and discharges were suppressed by nap(ts) in bss nap double mutants, whereas Sh heightened seizure susceptibility in bss Sh(133) and bss Sh(M) double mutants. Our results suggest that individual seizure repertoire components reflect different neural network activities that could be differentially altered by mutations of specific ion channel subunits.     

Ba2+-induced changes in the Na+- and K+-permeability of the isolated frog skin

Addition of the K+-channel blocking agent Ba2+ to the basolateral solution (in a concentration which is assumed to block the K+-flux via the K+-channels completely) resulted initially in a two-thirds reduction in the short-circuit current (SCC), followed by a complete recovery of the SCC. To examine the reason for this recovery, experiments were carried out which made it possible to calculate the Na+-permeability of the apical membrane (PaNa) and the K+-permeability of the basolateral membrane (PbK). The presence of Ba2+ had no significant effect on the cell volume and the cellular Na+- and K+-concentration. Addition of Ba2+ resulted in a depolarization of the intracellular potential (VSCC) from a control value of -76.3 +/- 2.8 mV to -15.1 +/- 1.7 mV. Although a complete recovery in the SCC was observed, VSCC did not recover. The K+-flux across the basolateral membrane was estimated from washout experiments. The washout of 42K+ (the K+-efflux) could be described by a single exponential component with a half time of 30-70 min. The addition of Ba2+ during the washout resulted in a transient decrease in 42K+-efflux from the epithelium. From VSCC and the cellular K+ and Na+-concentration and the coupling ratio of the Na-K pump, it was found that Na+-permeability of the apical membrane was 6.5 X 10(-7) cm X s-1 before the addition of Ba2+ and 1.7 X 10(-6) cm X s-1 when the SCC had recovered after the addition of Ba2+ and PbK changed from 8.8 X 10(-6) cm X s-1 to 1.5 X 10(-6) cm X s-1. Thus, the observed recovery in SCC was due to a considerable increase in Na+-permeability of the apical membrane and the presence or appearance of a small Ba2+-insensitive K+-permeability in the basolateral membrane.     

Inhibition of K(+)-evoked glutamate release from rat neocortical and hippocampal slices by gabapentin

Gabapentin (Neurontin((R))) has preclinical and clinical efficacy as an anticonvulsant, antihyperalgesic, anxiolytic, and neuroprotective drug. Since L-glutamic acid (GLU) is involved in various CNS (central nervous system) disorders, gabapentin may attenuate the release of this neurotransmitter possibly by interacting with the auxiliary alpha(2)delta subunit of voltage-sensitive calcium channels (VSCC). The effects of gabapentin, pregabalin (S-(+)-3-isobutylgaba) and its enantiomer R-(-)-3-isobutylgaba, and N- and P/Q-type VSCC-targeting peptide ligands (omega-conotoxin MVIIA, omega-conotoxin MVIIC, omega-agatoxin TK) were assessed in vitro on K(+)-evoked (endogenous) GLU release from rat neocortical and hippocampal slices. Gabapentin and pregabalin decreased GLU release by 11-26% with R-(-)-3-isobutylgaba being less effective than pregabalin. The reference N- and P/Q-type VSCC-targeting ligands reduced GLU release by 19-55% to implicate these VSCC in this Ca(2+)-dependent process. The inhibitory effect of gabapentin and related compounds on GLU release may reflect a subtle modulation of VSCC function which normalizes pathological changes in neurotransmitter release.     

Differential role of KIR channel and Na(+)/K(+)-pump in the regulation of extracellular K(+) in rat hippocampus.

Little information is available on the specific roles of different cellular mechanisms involved in extracellular K(+) homeostasis during neuronal activity in situ. These studies have been hampered by the lack of an adequate experimental paradigm able to separate K(+)-buffering activity from the superimposed extrusion of K(+) from variably active neurons. We have devised a new protocol that allows for such an analysis. We used paired field- and K(+)-selective microelectrode recordings from CA3 stratum pyramidale during maximal Schaffer collateral stimulation in the presence of excitatory synapse blockade to evoke purely antidromic spikes in CA3. Under these conditions of controlled neuronal firing, we studied the [K(+)]o baseline during 0.05 Hz stimulation, and the accumulation and rate of recovery of extracellular K(+) at higher frequency stimulation (1-3 Hz). In the first set of experiments, we showed that neuronal hyperpolarization by extracellular application of ZD7288 (11 microM), a selective blocker of neuronal I(h) currents, does not affect the dynamics of extracellular K(+). This indicates that the K(+) dynamics evoked by controlled pyramidal cell firing do not depend on neuronal membrane potential, but only on the balance between K(+) extruded by firing neurons and K(+) buffered by neuronal and glial mechanisms. In the second set of experiments, we showed that di-hydro-ouabain (5 microM), a selective blocker of the Na(+)/K(+)-pump, yields an elevation of baseline [K(+)]o and abolishes the K(+) recovery during higher frequency stimulation and its undershoot during the ensuing period. In the third set of experiments, we showed that Ba(2+) (200 microM), a selective blocker of inwardly rectifying K(+) channels (KIR), does not affect the posttetanus rate of recovery of [K(+)]o, nor does it affect the rate of K(+) recovery during high-frequency stimulation. It does, however, cause an elevation of baseline [K(+)]o and an increase in the amplitude of the ensuing undershoot. We show for the first time that it is possible to differentiate the specific roles of Na(+)/K(+)-pump and KIR channels in buffering extracellular K(+). Neuronal and glial Na(+)/K(+)-pumps are involved in setting baseline [K(+)]o levels, determining the rate of its recovery during sustained high-frequency firing, and determining its postactivity undershoot. Conversely, glial KIR channels are involved in the regulation of baseline levels of K(+), and in decreasing the amplitude of the postactivity [K(+)]o undershoot, but do not affect the rate of K(+) clearance during neuronal firing. The results presented provide new insights into the specific physiological role of glial KIR channels in extracellular K(+) homeostasis.     

Development of Na(+)- and K(+)-currents in the cochlear ganglion of the chick embryo.

The development of Na(+)- and K(+)-currents in the primary afferent neurons of the cochlear ganglion was studied using the patch-clamp technique. Cells were dissociated between days 6 and 17 of development and membrane currents recorded within the following 24 h. Outward currents were the first to appear between days 6 and 7 of embryonic development and their magnitude increased throughout development from 200 pA on day 7 to 900 pA on days 14-16. Threshold for activation decreased by 20 mV between days 8 and 14. Outward currents were absent when Cs+ replaced K+ in the pipette and were partially blocked by external tetraethylammonium. Outward currents contained at least three components: (i) a non-inactivating outward current, similar to the delayed-rectifier, predominating in mature neurons; (ii) a slowly inactivating current (tau about 200 ms), most evident in early and intermediate stages (days 7-10); and (iii) a rapidly inactivating outward current (tau about 20 ms) similar to the A-current (IA) described in other neurons, which was distinctly expressed in mature neurons. Sodium currents were identified as fast transient inward currents, sensitive to tetrodotoxin and extracellular Na(+)-removal. They appeared later than K(+)-currents and increased in size from about 100 pA between days 9-11 to 600 pA by days 13-16. The development of membrane currents in cochlear ganglion neurons corresponded to defined stages of the innervation pattern of the chick cochlea [Whitehead and Morest (1985) Neuroscience 14, 255-276]. These currents could be functionally related to the establishment of synaptic connections between transducing cells and primary afferent neurons.     

K(+)-induced changes in the properties of the hyperpolarization-activated cation current I(h) in rat CA1 pyramidal cells

The effects of rises in external K(+) (K(ext)) on I(h) were investigated in CA1 pyramidal cells of rat hippocampal slices using the whole-cell patch clamp technique. At the basal K(ext) level (2.5 mM), hyperpolarization-activated cation current (I(h)) had a maximal amplitude of -350+/-60 pA which was enhanced by approximately 60 and approximately 95% at 5 and 7.5 mM K(ext), respectively. The midpoint activation voltage was significantly shifted from -80 mV in the negative direction to about -87 mV at both 5 and 7.5 mM K(ext), without appreciable alterations of the current kinetics. The maximal conductance was approximately 2.4 nS under control conditions and significantly increased to approximately 3.3 and approximately 5.6 nS at 5 and 7.5 mM K(ext), respectively. The reversal potential was shifted in the positive direction, from a control value of approximately -30 mV by approximately 6 and approximately 14 mV at 5 and 7.5 mM K(ext), respectively. Our data demonstrate that even moderate changes in K(ext) have a substantial effect on the properties of I(h).     

Gentamicin inhibition of Na+,K(+)-ATPase in rat kidney cells.

Na,K(+)-ATPase activity is decreased in homogenized renal tissue from GM-treated rats. This study examines whether the site of the active effect of GM on Na,K(+)-ATPase activity in the kidney can be localized to the proximal convoluted tubules (PCT) where the drug is taken up and where it will produce necrosis. In rats treated with gentamicin (50 micrograms.kg-1.day-1 i.m.) for 7 days, PCT Na,K(+)-ATPase activity was reduced as compared to vehicle-treated rats but returned to control levels 7 days after treatment withdrawal. In another nephron segment, the medullary thick ascending limb of Henle (mTAL), where GM induced lesions are uncommon, Na,K(+)-ATPase activity was the same in GM- and vehicle-treated rats treatment. To study the in vitro effect of GM, dissected PCT and mTAL segments from untreated rats were preincubated for 30 min with GM 10(-3) M, a dose similar to the tissue concentration in chronically treated rats. In tubule segments that were permeabilized to allow the drug to enter the cells, GM 10(-3) M significantly inhibited Na,K(+)-ATPase activity both in PCT and mTAL. In non-permeabilized mTAL segments GM did not inhibit Na,K(+)-ATPase activity. GM inhibition of Na,K(+)-ATPase activity in permeabilized PCT segments persisted after the tubules were rinsed in GM free medium. GM does not inhibit Na,K(+)-ATPase partly purified from the renal cortex. Conclusion. Gentamicin inhibits Na,K(+)-ATPase activity in renal tubule cells when it has access to the cytoplasm. Treatment with GM will therefore cause a selective inhibition of Na,K(+)-ATPase in the proximal tubule cells.     

A test of K(+)-sensitive electrode responses to changes in K+ concentration

Tests of K(+)-sensitive microelectrodes were made using a plastic chamber, which consisted of upper and lower compartments separated by a partition with a small opening in the center. After the upper and lower compartments were filled with 25 and 100 mM KCl respectively, the tip of a K(+)-sensitive electrode was moved up and down through the partition opening, while recording potential changes. Some hydrodynamic events associated with electrode movement and effects of gravity upon spherical diffusion are discussed.     

Effects of cytotoxins on intracellular Na/K balance in mouse embryonic cell

Cytoplasmic Na/K imbalance induced by cytotoxic substances (ethylene glycol and cytochalasin B) was studied. Incubation in Dulbecco’s medium with these cytotoxins caused changes in K and Na concentrations in the mouse two-cell embryo blastomer. The effects of both substances resulted in a drop of Na content in the embryonic cell. Washing from cytochalasin B restored the initial Na/K balance in the cytoplasm. Possible adaptive mechanisms involved in the regulation of intracellular ionic homeostasis are discussed. __________ Translated from Kletochnye Tehnologii v Biologii i Meditsine, No. 2, pp. 102–105, April, 2008     

Stimulation of Xenopus oocyte Na(+),K(+)ATPase by the serum and glucocorticoid-dependent kinase sgk1

The serum and glucocorticoid-dependent kinase-1 (sgk1) is expressed in a wide variety of tissues including renal epithelial cells. As it is up-regulated by aldosterone, it is considered to participate in the regulation of renal Na(+) reabsorption. Indeed, co-expression of sgk1 with the renal epithelial Na(+) channel (ENaC) augments Na(+) channel activity. The aim of the present study was to examine possible effects of sgk1 on Na(+)/K(+)-ATPase activity. To this end dual-electrode voltage-clamp experiments were performed in Xenopus oocytes expressing the active kinase (S422D)sgk1 or the inactive mutant (K127N)sgk1. Na(+)/K(+)-ATPase activity was estimated from the hyperpolarization (delta V(m)) and the outwardly-directed current ( I(P)) created by addition of extracellular K(+) in the presence of K(+) channel blocker Ba(2+). Both delta V(m) and I(P) were significantly larger in oocytes expressing (S422D)sgk1 than in those expressing (K127N)sgk1 or having been injected with water. I(P) was fully inhibited by ouabain. Ion-selective microelectrodes showed that the stimulation of pump current was not the result of altered cytosolic Na(+) activity or pH. The present results thus point to an additional action of sgk1 that may participate in the regulation of renal tubular Na(+) transport. Moreover, sgk1 may be involved in the regulation of Na(+)/K(+)-ATPase in extrarenal tissues.     

Modulation of Na(+) x H(+) exchange by osmotic shock in isolated bovinearticular chondrocytes

The effects of hyperosmotic shock on intracellular pH (pHi) have been characterized in bovine articular chondrocytes. Osmotic shock is one of a variety of physicochemical stimuli experienced by chondrocytes upon cartilage loading. Cells were isolated from their extracellular matrix, and loaded with the pH-sensitive fluorophore 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. Hyperosmotic shocks were imposed by addition of KCl or sucrose to the extracellular medium. For cells at steady-state pHi, resuspension in hyperosmotic solutions elicited an alkalinization, which was significantly inhibited by removal of extracellular Na+ ions, or treatment with amiloride (1 mM) or HOE-694 (10 microM), both inhibitors of Na+ x H+ exchange. For cells acidified by ammonium rebound, recovery of pHi towards resting levels was significantly stimulated by exposure to hyperosmotic solutions, and the effect was again attenuated by inhibition of Na+ x H+ exchange. Determination of the rate of acid extrusion at different levels of acidification indicated that the affinity of acid extrusion systems for H+ ions was increased by hypertonic shock. The response to hyperosmotic media could be abolished by treatment of chondrocytes with the non-specific kinase inhibitor staurosporine (10 nM), while the phosphatase inhibitor okadaic acid (1 mM) was able to augment recovery rates to values similar to those measured under hyperosmotic conditions. The osmotic sensitivity of recovery was unaffected by exposure to the protein kinase C inhibitor calphostin C, but was abolished in cells treated with ML-7, a specific inhibitor of myosin light chain kinase. These results confirm that - as for other components of mechanical load - increased osmolarity can modulate the activity of Na+ x H+ exchange, in this case by altered patterns of phosphorylation of transporter-associated myosin. The changes of pHi which will result dictate in part the rate of cartilage macromolecule synthesis.     

Effects of electroconvulsive seizures on Na(+),K(+)-ATPase activity in the rat hippocampus

Although several advances have occurred concerning the use of electroconvulsive therapy, little progress has been made in understanding the mechanisms underlying its therapeutic or side effects. Na(+),K(+)-ATPase is an important enzyme of central nervous system, responsible for ionic gradient maintenance and consumption of approximately 40-50% of brain ATP. This work was performed in order to determine Na(+),K(+)-ATPase activity after acute and chronic electroconvulsive shock. Results showed an inhibition of Na(+),K(+)-ATPase activity in the hippocampus 48 h, 7, 30, 60 and 90 days after a single electroconvulsive shock. Chronic treatment diminished the enzyme activity in the hippocampus 7 and 30 days after electroconvulsive (ECS) sessions. Our findings demonstrated that Na(+),K(+)-ATPase activity is altered by ECS.     

Inhibition of Na+-dependent GABA uptake in isolated frog sensory nerve cell bodies by extra- and intracellular Li+

The effect of Li+ on the Na+-dependent gamma-aminobutyric acid (GABA) uptake was investigated by examining the effects of external and internal Li+ ([Li]o and [Li]i) on GABA-gated Cl- current (ICl) in the frog dorsal root ganglion cells using the suction pipette technique which allows the internal perfusion under voltage-clamp condition. The suppression of GABA responses in the presence of external Na+ ([Na]o) was larger at lower than at higher GABA concentration. Replacement of [Na]o with Li+ completely removed the Na+ suppression, and GABA dose-response curve in Li+ external solution agreed well with that in Na+-free (Tris+) external solution. Either increasing [Li]i) or internal Na+ ([Na]i) at a constant [Na]o equally reduced the Na+-dependent suppression of GABA-gated ICl. The results indicate that both the [Li]o and [Li]i remove in different manner the [Na]o-dependent suppression of GABA-induced ICl:i.e. the [Li]o acts as a blocker of Na+-GABA co-transport mechanism while the increase of [Li]i decreases the Na+ electrochemical potential gradient across the soma membrane as well as [Na]i does.