Rise In Na Research Articles

5‐hydroxytryptamine‐mediated increase in glutamate uptake by the leech giant glial cell

The clearance of synaptically released glutamate is one of the pivotal functions of glial cells. We have studied the role of 5-hydroxytryptamine (5-HT, 30 microM), a neurotransmitter and neurohormone in the leech central nervous system with a versatile action spectrum, on the efficacy of glial glutamate uptake. The activity of the glutamate uptake carrier in the giant glial cell in isolated ganglia of Hirudo medicinalis was monitored by measuring the membrane current and the change in the intracellular Na(+) concentration (Na(+) (i)) as induced by the glutamate carrier substrate D-aspartate (D-asp, 1 mM). 5-HT increased the D-asp-induced current (EC(50) at 5 microM) and rise in Na(+) (i), an effect which was mimicked by the membrane-permeable cyclic nucleotide analogue dibutyryl-cyclic AMP (db-cAMP). The adenylyl cyclase inhibitor SQ 22,536 and the protein kinase A antagonist Rp-cAMP inhibited the effect of 5-HT. Blocking the G protein in the giant glial cell by injecting GDP-beta-S suppressed the effect of 5-HT, but not the effect of db-cAMP, on the D-asp-induced current. Our results suggest that 5-HT enhances the glial uptake of glutamate via cAMP- and PKA-mediated pathway.

Managing hyperosmolar hyperglycaemic syndrome

I would like to comment on the Association of British Clinical Diabetologists (ABCD) guidelines for the management of hyperglycaemic emergencies in adults (Practical Diabetes International June 2006, pages 227-231). Although the guidelines are simply and straightforwardly presented, I feel they seriously underplay the severity of hyperosmolar nonketotic coma (which incidentally is a misleading term: it should be abandoned and replaced by hyperosmolar hyperglycaemic syndrome, HHS). I think it is a mistake to describe the management ‘as for DKA [diabetic ketoacidosis] but …’ as there are some significant differences which need to be emphasised. Failure to understand the underlying disorder may lead to inappropriate management. There is no precise or internationally agreed definition of HHS but there is recognition that minor degrees of ketosis and acidosis (arterial pH >7.3, bicarbonate >15mmol/L) are common. Patients with HHS are often elderly and the condition may be the first presentation of type 2 diabetes. It appears to be more common in people of African or Afro-Caribbean origin. In recent years, there have been case reports of HHS occurring in children and young adults — not all with type 2 diabetes. Severe dehydration and pre-renal failure are usual. Unlike DKA, the onset is over many days and, although it is correct to say that vascular complications are common, cerebral oedema is the most serious complication and may present as seizures, progressive confusion, or altered consciousness and lead to permanent neurological damage or death. Correct interpretation of the serum Na is important. Total body Na is low and there may be marked water deficiency. Although the initial Na may be normal or high, it is important to understand that high concentrations of effective solutes such as glucose will cause fluid shifts out of the cell and a dilutional fall in Na. The corrected Na can be calculated by adding 2.4mmol/L for every 5.5mmol/L where the glucose is above normal but, more importantly, it follows that as treatment results in a fall in glucose, there will almost inevitably be a rise in serum Na. Therefore, the rise in Na is expected and predictable — what is important is what is happening to tonicity. Thus, a rising Na alone is not an indication for the administration of hypotonic fluids. Care must be taken to avoid situations which may increase the risk of cerebral oedema. Extracellular hypertonicity causes cellular dehydration which in the brain is resisted by a protective mechanism known as cerebral volume regulation. Water loss is limited by the production of intracellular osmotically active ions and, later, organic osmolytes or idiogenic osmoles. Whilst this rise in cellular osmolality is beneficial in the hyperosmolar state, rapid rehydration with IV fluids may lead to water movement into the cell and swelling occurs before compensatory mechanisms have time to eliminate these solutes. This may be the mechanism which leads to cerebral oedema and is the reason why many argue that hypotonic solutions should be avoided (there is no experimental evidence to justify using less than 0.5N saline) and that slower correction of the hyperosmolality is safer than rapid correction. Assessing the level of dehydration is difficult and often over-estimated. Postural changes in heart rate and BP can be helpful but not always practical. It is rare to need more than twice the usual daily fluid requirements. The aim is to replace approximately 50% of the deficit in the first 12 hours. These patients are often transferred too soon to general medical wards and fluid management left to inexperienced junior medical staff. Severe HHS should be seen soon after admission by diabetes specialists with experience of managing this challenging emergency, and close supervision given until the patient is eating and drinking normally. Adrian Scott*, * Diabetes Centre, Northern General Hospital, Herries Road, Sheffield S5 7AU, UK

Single and combined effects of carbamazepine and vinpocetine on depolarization-induced changes in Na +, Ca 2+ and glutamate release in hippocampal isolated nerve endings

The single and combined effects of carbamazepine and vinpocetine on the release of the excitatory amino acid neurotransmitter glutamate, on the rise in internal Na + (Na i, as determined with SBFI), and on the rise in internal Ca 2+ (Ca i, as determined with fura-2) induced by an increased permeability of presynaptic Na + channels, with veratridine, or by an increased permeability of presynaptic Ca 2+ channels with high K +, were investigated in isolated hippocampal nerve endings. The present study shows that carbamazepine and vinpocetine, both inhibit dose dependently the release of preloaded [ 3H]Glu induced by veratridine. However, carbamazepine is two orders of magnitude less potent than vinpocetine. The calculated IC 50's for carbamazepine and vinpocetine to inhibit veratridine-induced [ 3H]Glu release are 200 and 2 μM, respectively. Consistently 150 μM carbamazepine and 1.5 μM vinpocetine reduce the veratridine-induced rise in Na i in a similar extent. The single effects of carbamazepine and of vinpocetine on the presynaptic Na + channel mediated responses, namely the rise in Na i and the release of Glu induced by veratridine, are additive. Responses that depend on the entrance of external Ca 2+ via presynaptic Ca 2+ channels, such as the release of [ 3H]Glu and the rise in Ca i induced by high K +, are insensitive to 300 μM carbamazepine and slightly reduced by 5 μM vinpocetine. It is concluded that the additive effects of carbamazepine, which is one of the most common antiepileptic drugs, and vinpocetine that besides its known neuroprotective action and antiepileptic potential is a memory enhancer, may perhaps be advantageous in the treatment of epileptic patients.

Vinpocetine blockade of sodium channels inhibits the rise in sodium and calcium induced by 4-aminopyridine in synaptosomes

The objective of this study was to get a more understandable picture of the mechanism underlying the anticonvulsant action of vinpocetine. The question of how the cerebral excitability is affected was investigated by determining the effect of vinpocetine on the changes on the internal concentrations of Na + (Na i ) and Ca 2+ (Ca i ) induced by different concentrations of the convulsing agent 4-aminopyridine (4-AP) in striatal isolated nerve endings. The cytosolic concentrations of Na i and Ca i were detected fluorimetrically with sodium-binding benzofuran isophthalate (SBFI) and fura-2, respectively. Vinpocetine, like the Na + channel blocker, tetrodotoxin, abolished the increase in Na i induced by 0.1 mM 4-AP and only inhibited in 30% the rise in Na i induced by 1 mM 4-AP. In contrast with the different sensitivity of the rise in Na i induced by 0.1 and 1 mM 4-AP to vinpocetine and tetrodotoxin, the rise in Ca i induced by the two concentrations of 4-AP was markedly inhibited by vinpocetine (and tetrodotoxin), indicating that only the voltage-sensitive sodium channels (VSSC)-mediated fraction of the rise in Na i induced by 4-AP is linked with the activation of pre-synaptic Ca 2+ channels. The elevation of Ca 2+ induced by high K + (30 mM) does not require a Na + gradient and is vinpocetine and tetrodotoxin insensitive. In contrast, the elevation of Ca i induced by 4-AP, requires a physiological (out/in) Na + gradient and is vinpocetine and tetrodotoxin-sensitive. It is concluded that by blocking the tetrodotoxin-sensitive fraction of the rise in Na i induced by 4-AP, vinpocetine inhibits the concomitant rise in Ca i induced by 4-AP. The inhibitory effect of vinpocetine on pre-synaptic voltage-sensitive sodium channels may underlie the in vivo anticonvulsant action of vinpocetine.

Ouabain‐induced reversal of astrocytic GLT‐1 crucial to neuronal death in neuron/astrocyte co‐cultures

AbstractWe investigated whether the role of the astrocytic glutamate transporter GLT‐1 is reversed when the ionic gradient across plasma membrane has collapsed, and if so, whether the reversal is crucial to neuronal death. To estimate the direction of glutamate transport by GLT‐1, Na+ movement coupled with glutamate transport in astrocytes was analyzed in mixed astrocyte/neuron cultures. The rise in astrocytic intracellular Na+ due to glutamate exposure was suppressed by co‐treatment with dihydrokainate (DHK). In contrast, the ouabain‐induced rise in Na+ was significantly enhanced by DHK, suggesting that the role of GLT‐1 was reversed. We then analyzed whether the reversal increased the amount of extracellular glutamate, and was crucial to excitotoxic neuronal death. Ouabain treatment resulted in a marked rise in glutamate and in neuronal death, and both the rise and cell death were significantly attenuated by DHK treatment.

A regulated apical Na(+) conductance in dexamethasone-treated H441 airway epithelial cells.

Treating H441 cells with dexamethasone raised the abundance of mRNA encoding the epithelial Na(+) channel alpha- and beta-subunits and increased transepithelial ion transport (measured as short-circuit current, I(sc)) from <4 microA.cm(-2) to 10-20 microA.cm(-2). This dexamethasone-stimulated ion transport was blocked by amiloride analogs with a rank order of potency of benzamil >or= amiloride > EIPA and can thus be attributed to active Na(+) absorption. Studies of apically permeabilized cells showed that this increased transport activity did not reflect a rise in Na(+) pump capacity, whereas studies of basolateral permeabilized cells demonstrated that dexamethasone increased apical Na(+) conductance (G(Na)) from a negligible value to 100-200 microS.cm(-2). Experiments that explored the ionic selectivity of this dexamethasone-induced conductance showed that it was equally permeable to Na(+) and Li(+) and that the permeability to these cations was approximately fourfold greater than to K(+). There was also a small permeability to N-methyl-d-glucammonium, a nominally impermeant cation. Forskolin, an agent that increases cellular cAMP content, caused an approximately 60% increase in I(sc), and measurements made after these cells had been basolaterally permeabilized demonstrated that this response was associated with a rise in G(Na). This cAMP-dependent control over G(Na) was disrupted by brefeldin A, an inhibitor of vesicular trafficking. Dexamethasone thus stimulates Na(+) transport in H441 cells by evoking expression of an amiloride-sensitive apical conductance that displays moderate ionic selectivity and is subject to acute control via a cAMP-dependent pathway.

Characterization of the participation of sodium channels on the rise in Na+ induced by 4-aminopyridine (4-AP) in synaptosomes.

The participation of voltage-sensitive Na+ channels (VSSC) on the changes on internal (i) Na+, K+, Ca2+, and on DA, Glu, and GABA release caused by different concentrations of 4-AP was investigated in striatum synaptosomes. TTX, which abolished the increase in Na(i) (as determined with SBFI), induced by 0.1 mM 4-AP only inhibited by 30% the rise in Na(i) induced by 1 mM 4-AP. One millimolar 4-AP markedly decreased the fluorescence of the K+ indicator dye PBFI but 0.1 mM 4-AP did not. Like 1 mM 4-AP, ouabain decreased PBFI fluorescence and increased a considerable fraction of Na(i) in a TTX-insensitive manner. In contrast with the different TTX sensitivity of the rise in Na(i) induced by 0.1 and 1 mM 4-AP, the rise in Ca(i) (as determined with fura-2) induced by the two concentrations of 4-AP was markedly inhibited by TTX, as well as by omega-agatoxin in combination with omega-conotoxin GVIA, indicating that only the TTX-sensitive fraction of the rise in Na(i) induced by 4-AP is linked with the activation of presynaptic Ca2+ channels. It is concluded that the TTX-sensitive fraction of neurotransmitter release evoked by 4-AP is released by exocytosis, and the TTX insensitive fraction involves reversal of the neurotransmitters transporters. This contrasts with the exocytosis evoked by high K+ that is unchanged by TTX and with the neurotransmitter-transporter-mediated release evoked by veratridine, which is highly TTX sensitive and does not require activation of Ca2+ channels.

R 56865 exerts cardioprotective properties independent of the intracellular Na(+)-overload in the guinea pig heart.

Reducing intracellular Na(+)-accumulation during ischemia exerts cardioprotective effects in reperfusion in a variety of models. Since slowly-inactivating Na(+)-channels may contribute to Na(+)-influx in ischemia, we investigated whether the ischemia-protective properties of R 56865, an inhibitor of slowly inactivating Na(+)-channels, are mediated by inhibition of the ischemic Na(+)-overload. Monitoring intracellular Na(+) (Na(+)(i)) by (23)Na-NMR-spectroscopy revealed a continuous rise of Na(+)(i) during ischemia in the isolated perfused guinea pig heart. Within 30 and 60 min of ischemia, respectively, Na(+)(i) had risen 2.6+/-0.2- and 4.4+/-0.2-fold compared to baseline ( n=6). R 56865 (1 microM) did not influence the time course of the Na(+)(i)-accumulation at any point of the ischemic period. R 56865, however, showed marked cardioprotective properties: in the reperfusion period the agent markedly improved the restoration of left ventricular developed pressure (29.1+/-6.8 mm Hg vs. 2.4+/-2.0 mm Hg), ATP (2.8+/-0.3 mM vs. 1.7+/-0.6 mM) and phosphocreatine (10.9+/-2.2 mM vs. 6.8+/-1.1 mM), furthermore contracture development was reduced. The present study strongly suggests slowly-inactivating Na(+)-channels being at best a minor port of Na(+)-entry in the ischemic guinea pig heart. It clearly demonstrates that the potent cardioprotective properties of R 56865 are unrelated to intracellular sodium homeostasis.

Hypotonic stimulation of the Na+ active transport in frog skeletal muscle: role of the cytoskeleton

Hypotonicity produces a marked activation of the Na+ pump in frog sartorius muscle. The increase in net Na+ efflux under hypotonic conditions occurs despite the reductions in [Na+]i that are due to fibre swelling and Na+ loss. The pump density (ouabain binding) increases not only upon reduction of the medium osmotic pressure (π) from its normal value (π= 1) to one-half (π= 0.5), but also in muscles that are returned to π= 1 after equilibration in π= 2 medium. The equilibration in π= 2 medium does not affect pump density. Ouabain-binding increments cannot be ascribed to a rise in the Na+–K+ exchange rate of a fixed number of pumps: they also occurred in the continued presence of a saturating concentration of ouabain (50 μm). Under those conditions, the π= 1 ★π= 0.5 transfer produced a 43 % increase in pump sites, while the π= 2 ★π= 1 transfer induced a rise of 46 %. Actinomycin D did not alter the stimulation of Na+ extrusion elicited by hypotonicity, suggesting that de novo synthesis of pumps was not involved in the increase of the apparent number of pump sites. Disruption of microtubules by colchicine (100 μm) and intermediate filaments by acrylamide (4 mm) did not alter the hypotonic effect. Likewise, genistein (100 μm), a specific inhibitor of tyrosine kinase, did not affect significantly the hypotonic response. Microfilament-disrupting agents like cytochalasin B (5 μm) and latrunculin B (10 μm) reduced the increase in Na+ efflux induced by π= 1 ★π= 0.5 transfer by about 35 % and 72 %, respectively. Latrunculin B reduced the increases in pump density generated by π= 1 ★π= 0.5 and π= 2 ★π= 1 transfers by about 79 % and 91 %, respectively. The results suggest that the membrane stretch due to hypotonic fibre volume increase would promote a microfilament-mediated insertion of submembranous spare Na+ pumps in the sarcolemma and, consequently, the rise in active Na+ transport.

Intracellular Na+ regulation in cardiac myocytes.

Intracellular [Na+] ([Na+]i) is regulated in cardiac myocytes by a balance of Na+ influx and efflux mechanisms. In the normal cell there is a large steady state electrochemical gradient favoring Na+ influx. This potential energy is used by numerous transport mechanisms, including Na+ channels and transporters which couple Na+ influx to either co- or counter-transport of other ions and solutes. Six sarcolemmal Na+ influx pathways are discussed in relatively quantitative terms: Na+ channels, Na+/Ca2+ exchange, Na+/H+ exchange, Na+/Mg2+ exchange, Na+/HCO3- cotransport and Na+/K+/2Cl- cotransport. Under normal conditions Na+/Ca2+ exchange and Na+ channels are the dominant Na+ influx pathways, but other transporters may become increasingly important during altered conditions (e.g. acidosis or cell volume stress). Mitochondria also exhibit Na+/Ca2+ antiporter and Na+/H+ exchange activity that are important in mitochondrial function. These coupled fluxes of Na+ with Ca2+, H+ and HCO3- make the detailed understanding of [Na+]i regulation pivotal to the understanding of both cardiac excitation-contraction coupling and pH regulation. The Na+/K+-ATPase is the main route for Na+ extrusion from cells and [Na+]i is a primary regulator under physiological conditions. [Na+]i is higher in rat than rabbit ventricular myocytes and the reason appears to be higher Na+ influx in rat with a consequent rise in Na+/K+-ATPase activity (rather than lower Na+/K+-ATPase function in rat). This has direct functional consequences. There may also be subcellular [Na+]i gradients locally in ventricular myocytes and this may also have important functional implications. Thus, the balance of Na+ fluxes in heart cells may be complex, but myocyte Na+ regulation is functionally important and merits focused attention as in this issue.

Increased Na+ concentration and altered Na/K pump activity in hypertrophied canine ventricular cells.

To investigate whether hypertrophy in the dog with chronic atrioventricular block (CAVB) alters [Na+]i and Na/K-pump function of ventricular myocytes. We measured the [Na+]i dependence of the Na/K pump current, I(p). This relation was used as a calibration curve for [Na+]i based on I(p). We measured I(p) at the time of access and extrapolated [Na+] at the pump sites, i.e. subsarcolemmal [Na+], [Na+](subs), from the calibration curve. The extrapolated [Na+](subs) was significantly higher in CAVB (7.9 vs. 3.2 mM in control). The [Na+]i dependence of I(p) in CAVB myocytes was shifted to the right (range of [Na+](i): 0-20 mM). In resting cells, the I(p), i.e. steady state Na+ efflux, which matches Na+ influx, was higher in CAVB (0.25+/-0.02 vs. 0.47+/-0.06 pA/pF, P<0.05). Maximal I(p) density was not different, and DHO sensitivity was not altered. Hypertrophy in CAVB cells is associated with increased [Na+](subs). This results from an increase in Na+ influx, and a decreased sensitivity of I(p) for Na+ in the range of [Na+]i studied. There is no evidence for a decrease in total pump capacity or for a functional Na/K-ATPase isoform shift. The rise in Na+ contributes to the contractile adaptation and preservation of sarcoplasmic reticulum Ca2+ content at the low heart rates of the dog with CAVB.

Hypotonic stimulation of the Na+ active transport in frog skeletal muscle: role of the cytoskeleton.

Hypotonicity produces a marked activation of the Na+ pump in frog sartorius muscle. The increase in net Na+ efflux under hypotonic conditions occurs despite the reductions in [Na+]i that are due to fibre swelling and Na+ loss. The pump density (ouabain binding) increases not only upon reduction of the medium osmotic pressure (pi) from its normal value (pi = 1) to one-half (pi = 0.5), but also in muscles that are returned to pi = 1 after equilibration in pi = 2 medium. The equilibration in pi = 2 medium does not affect pump density. Ouabain-binding increments cannot be ascribed to a rise in the Na+-K+ exchange rate of a fixed number of pumps: they also occurred in the continued presence of a saturating concentration of ouabain (50 microM). Under those conditions, the pi = 1 pi = 0.5 transfer produced a 43 % increase in pump sites, while the pi = 2 pi = 1 transfer induced a rise of 46 %. Actinomycin D did not alter the stimulation of Na+ extrusion elicited by hypotonicity, suggesting that de novo synthesis of pumps was not involved in the increase of the apparent number of pump sites. Disruption of microtubules by colchicine (100 microM) and intermediate filaments by acrylamide (4 mM) did not alter the hypotonic effect. Likewise, genistein (100 microM), a specific inhibitor of tyrosine kinase, did not affect significantly the hypotonic response. Microfilament-disrupting agents like cytochalasin B (5 microM) and latrunculin B (10 microM) reduced the increase in Na+ efflux induced by pi = 1 pi = 0.5 transfer by about 35 % and 72 %, respectively. Latrunculin B reduced the increases in pump density generated by pi = 1 pi = 0.5 and pi = 2 pi = 1 transfers by about 79 % and 91 %, respectively. The results suggest that the membrane stretch due to hypotonic fibre volume increase would promote a microfilament-mediated insertion of submembranous spare Na+ pumps in the sarcolemma and, consequently, the rise in active Na+ transport.

Electron theory study on mechanism of action of cobalt in Fe-Co-Cr based high-alloy steel

The valence electron structure of martensite in FeCoCr based high-alloy steel is calculated using the empirical electron theory of solids and molecules (EET). The results show that the incorporation of cobalt leads to a rise in nA from 0.3835 to 0.4684, which enhances the bonding forces between atoms in α-Fe matrix. Meanwhile, the incorporation of Co changes the valence electron structure of segregated structure units formed by C and other alloying elements, and increases nA for the segregated units containing C-Me significantly, resulting in changing the precipitation behavior of the secondary phases during tempering and strengthening the resistance to tempering.

CAMP and sodium transport in the freshwater crayfish, Cherax destructor

Crayfish in which sodium absorption was maximally stimulated had elevated levels of both cAMP and Na +-K +-ATPase activity in gill tissue. The concentration of cAMP and activity of Na +-K +-ATPase in gill tissue were monitored following transfer of crayfish from water containing 125 mmol.l −1 Na to Na-free media. Both parameters were significantly elevated within 10 min of transfer to Na-free media and [cAMP] peaked between 1 and 2 h before falling transiently to the control level at 3 h. A second peak of [cAMP] and a further rise in Na +-K +-ATPase activity were evident 6 h after transfer and elevated levels were then maintained. The pattern observed was consistent with the existence of two separate mechanisms for the control of sodium absorption both of which stimulated the activity of Na +-K +-ATPase via elevation of the intracellular concentration of cAMP. The initial response was very rapid (<10 min) but of brief duration (1–2 h) and this mechanism appeared to be sensitive to changes in external ion levels. The second mechanism exhibited a much longer response time (3–6 h) and duration and was likely to be sensitive to changes in internal ion concentrations.

Oxygen-evoked Na+ transport in rat fetal distal lung epithelial cells.

Monolayer cultures of rat fetal distal lung epithelial (FDLE) cells generated larger spontaneous short circuit currents (ISC) when maintained (48 h) at neonatal alveolar PO2 (100 mmHg) than at fetal PO2 (23 mmHg). When cells were shifted between these atmospheres in order to impose a rise in PO2 equivalent to that seen at birth, no rise in ISC was seen after 6 h but the response was fully established by 24 h. Studies of basolaterally permeabilised cells revealed a small rise in apical Na+ conductance (GNa) 6 h after PO2 was raised but no further change had occurred by 24 h. A substantial rise was, however, seen after 48 h. Reporter gene assays showed that no activation of the -ENaC (epithelial Na+ channel -subunit) promoter was discernible 24 h after PO2 was raised but increased transcriptional activity was seen at 48 h. Studies of apically permeabilised cells showed that a small rise in Na+ pump capacity was evident 6 h after PO2 was raised and, in common with the rise in ISC, this effect was fully established by 24 h. The rise in ISC thus develops 6-24 h after PO2 is raised and is due, primarily, to increased Na+ pump capacity. The increase in GNa thus coincides with activation of the -ENaC promoter but these effects occur after the rise in ISC is fully established and so cannot underlie this physiological response. The increased transcription may be an adaptation to increased Na+ transport and not its cause.

Activation of protein kinase A modulates trafficking of the human cardiac sodium channel in Xenopus oocytes.

Voltage-gated Na(+) channels are critical determinants of electrophysiological properties in the heart. Stimulation of beta-adrenergic receptors, which activate cAMP-dependent protein kinase (protein kinase A [PKA]), can alter impulse conduction in normal tissue and promote development of cardiac arrhythmias in pathological states. Recent studies demonstrate that PKA activation increases cardiac Na(+) currents, although the mechanism of this effect is unknown. To explore the molecular basis of Na(+) channel modulation by beta-adrenergic receptors, we have examined the effects of PKA activation on the recombinant human cardiac Na(+) channel, hH1. Both in the absence and the presence of hbeta(1) subunit coexpression, activation of PKA caused a slow increase in Na(+) current that did not saturate despite kinase stimulation for 1 hour. In addition, there was a small shift in the voltage dependence of channel activation and inactivation to more negative voltages. Chloroquine and monensin, compounds that disrupt plasma membrane recycling, reduced hH1 current, suggesting rapid turnover of channels at the cell surface. Preincubation with these agents also prevented the PKA-mediated rise in Na(+) current, indicating that this effect likely resulted from an increased number of Na(+) channels in the plasma membrane. Experiments using chimeric constructs of hH1 and the skeletal muscle Na(+) channel, hSKM1, identified the I-II interdomain loop of hH1 as the region responsible for the PKA effect. These results demonstrate that activation of PKA modulates both trafficking and function of the hH1 channel, with changes in Na(+) current that could either speed or slow conduction, depending on the physiological circumstances.

Synthesis of gill Na(+)-K(+)-ATPase in Atlantic salmon smolts: differences in alpha-mRNA and alpha-protein levels.

Several parameters were analyzed to determine the mechanisms responsible for the enhancement of the gill Na(+)-K(+)-ATPase activity of Atlantic salmon smolts. A major alpha-subunit transcript of 3.7 kb was revealed by Northern blot in both parr and smolt gills when hybridized with two distinct cDNA probes. The alpha-mRNA abundance demonstrated an increase to maximal levels in smolts at an early stage of the parr-smolt transformation. This was followed by a gradual rise in alpha-protein levels, revealed by Western blots with specific antibodies and by an increase in gill Na(+)-K(+)-ATPase hydrolytic activity, both only reaching maximum levels a month later, at the peak of the transformation process. Parr fish experienced a decrease in alpha-mRNA abundance and had basal levels of alpha-protein and enzyme activity. Measurement of the binding of [(3)H]ouabain to Na(+)-K(+)-ATPase was characterized in smolts and parr gill membranes showing more than a twofold elevation in smolts and was of high affinity in both groups (dissociation constant = 20-23 nM). Modulation of the enzyme due to increased salinity was also observed in seawater-transferred smolts, as demonstrated by an increase in alpha-mRNA levels after 24 h with a rise in Na(+)-K(+)-ATPase activity occurring only after 11 days. No qualitative change in alpha-expression was revealed at either the mRNA or protein level. Immunological identification of the alpha-protein was performed with polyclonal antibodies directed against the rat alpha-specific isoforms, revealing that parr, freshwater, and seawater smolts have an alpha(3)-like isoform. This study shows that the increase in Na(+)-K(+)-ATPase activity in smolt gills depends first on an increase in the alpha-mRNA expression and is followed by a slower rise in alpha-protein abundance that eventually leads to a higher synthesis of Na(+)-K(+) pumps.

Novel Effect of Insulin: Insulin-Stimulated Na+Transport Is Mediated by Hydrolysis of Phosphoinositides

Previous studies showed that insulin stimulation of electrogenic Na+transport in renal epithelial cells is mediated by a calcium-dependent signal transduction mechanism. The present study was performed to determine whether the insulin-induced increase in intracellular Ca2+(Ca2+i) was mediated by hydrolysis of phosphatidylinositol and release of inositol trisphosphate. Experiments were conducted with cultured A6 cells, derived fromXenopus laevis,grown on permeable supports. Addition of insulin resulted in 2 to 3 fold increases in inositol trisphosphate and a 50% increase in 1,2 diacylglycerol within 10s, which corresponded to the time-course, previously reported, of insulin stimulated increases in Na+transport and Ca2+i. Further studies showed that aldosterone, previously shown to stimulate an increase in 1,4,5-inositol trisphosphate at onset of the rise in Na+transport, also increased DAG levels during the initial phase of stimulation of Na+transport. These studies provide the first evidence that a biological response induced by insulin is mediated by hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) which results in two products, inositol trisphosphate which causes the release of Ca2+from intracellular stores and 1,2 diacylglycerol. In addition this study provides further support for the proposal that a common signal transduction mechanism mediates electrogenic Na+transport by multiple agonists.

Angiotensin II and angiotensin-(1-7) effects on free cytosolic sodium, intracellular pH, and the Na(+)-H+ antiporter in vascular smooth muscle.

The aim of the present study was to define the effects of angiotensin II (Ang II) and Ang-(1-7) on free cytosolic Na+ (Na+i), intracellular pH (pHi), and the Na(+)-H+ antiporter in cultured vascular smooth muscle cells from rat aorta. Cells were loaded with either BCECF-AM or SBFI-AM for measurement of pHi and Na+i, respectively. Ang II (10(-6) mol/L) caused a rapid rise in Na+i followed by a progressive increase that peaked at about 10 minutes (from 11 +/- 1.5 to 16 +/- 1.5 mmol/L, P < .001), whereas Ang-(1-7) (10(-6) mol/L) did not affect Na+i significantly (from 11.5 +/- 1.1 to 11.8 +/- 0.07 mmol/L). The effect of Ang II on Na+i was concentration dependent (delta Na+i, 5.1 +/- 0.9, 3.8 +/- 0.6, 1.6 +/- 0.6, and 0.14 +/- 0.18 mmol/L with decreasing concentrations of 10(-6), 10(-7), 10(-8), and 10(-9) mol/L, respectively). Ang II caused a brief acidification followed by an increase in pHi (from 7.34 +/- 0.03 to 7.43 +/- 0.03 after 10 minutes, P < .005), and Ang-(1-7) had no significant effect on pHi (from 7.23 +/- 0.03 to 7.23 +/- 0.03). To investigate whether pHi and Na+i changes induced by Ang II were due to cell Na+ entry via stimulation of the Na(+)-H+ antiporter, we pretreated cells with EIPA (25 mumol/L) or ouabain (2.0 mmol/L). Ang II in the presence of ouabain caused a greater increase than that seen with ouabain alone (delta Na+i, 13 +/- 1.5 versus 6.3 +/- 1.2 mmol/L, P < .0025). EIPA by itself decreased Na+i and pHi. After EIPA, Ang II failed to increase both Na+i and pHi, demonstrating that the Na(+)-H+ antiporter is responsible for the rises in Na+i and pHi during stimulation with Ang II. To further characterize the mechanism of Ang II action, we exposed cells to an Ang II type I receptor antagonist (L-158,809, 10(-6) mol/L) or two different type 2 receptor antagonists (PD 123177 and CGP 421112A, 10(-6) mol/L). L-158,809 completely blocked the rise in pHi caused by Ang II, whereas PD 123177 and CGP 421112A did not. We conclude that Ang II increases both Na+i and pHi, and both effects are mediated by stimulation of the Na(+)-H+ antiporter. Ang-(1-7), by contrast, has no significant effect on Na+i, pHi, or the Na(+)-H+ antiporter. Stimulation of this antiporter by Ang II is exerted through the type 1 receptor.

Delayed increase in intracellular Na+ in cerebral cortical slices during severe hypoxia as measured by double quantum filtered 23Na+ NMR.

We have used double quantum filtered (DQF) 23Na+ nuclear magnetic resonance (NMR) spectroscopy without shift reagents in order to monitor intracellular Na+ (Na+i) in a cortical brain slice preparation. The external Na+ (Na+o) signal was reduced by 95% by the DQF sequence compared with the directly observed 23Na+. The DQF 23Na+ signal is not exclusively due to Na+i, however, as 40% of this signal appears to arise from Na(+)-ions interacting with extracellular membrane proteins or proteins exposed at the cut surfaces of the slices. Veratridine increased instantly the DQF 23Na+ signal so that it reached 130.4 +/- 5.0% by 12 min. This shows that there was a significant contribution from Na+i in the DQF 23Na+ NMR spectra. Hypoxia of 30 min duration in the presence of 10 nM glucose did not influence intensity of the DQF 23Na+ signal. Aglycaemic hypoxia caused complete collapse of phosphocreatine (PCr) within 7 min whereas DQF 23Na+ first increased 15 min after the insult. This increase reached its maximal value of 125% after 25 min. There was an incomplete recovery of the DQF 23Na+ after aglycaemic hypoxia to 110% of the control value parallel to poor metabolic recovery. The presence of 10 mM extracellular Mg2+ had no apparent effect on the aglycaemic hypoxia-induced rise in Na+i indicating that it was linked to Ca2+ influx. Tetrodotoxin (TTx, 4.7 microM) did not influence the rise of Na+i caused by aglycaemic hypoxia. These results indicate that elevation of Na+i is a late consequence of energy failure in the cerebral cortex.