Accumulation Of Intracellular Na Research Articles

Primary Role of the Kidney in Pathogenesis of Hypertension.

Previous transplantation studies and the concept of 'nephron underdosing' support the idea that the kidney plays a crucial role in the development of essential hypertension. This suggests that there are genetic factors in the kidney that can either elevate or decrease blood pressure. The kidney normally maintains arterial pressure within a narrow range by employing the mechanism of pressure-natriuresis. Hypertension is induced when the pressure-natriuresis mechanism fails due to both subtle and overt kidney abnormalities. The inheritance of hypertension is believed to be polygenic, and essential hypertension may result from a combination of genetic variants that code for renal tubular sodium transporters or proteins involved in regulatory pathways. The renin-angiotensin-aldosterone system (RAAS) and sympathetic nervous system (SNS) are the major regulators of renal sodium reabsorption. Hyperactivity of either the RAAS or SNS leads to a rightward shift in the pressure-natriuresis curve. In other words, hypertension is induced when the activity of RAAS and SNS is not suppressed despite increased salt intake. Sodium overload, caused by increased intake and/or reduced renal excretion, not only leads to an expansion of plasma volume but also to an increase in systemic vascular resistance. Endothelial dysfunction is caused by an increased intracellular Na+ concentration, which inhibits endothelial nitric oxide (NO) synthase and reduces NO production. The stiffness of vascular smooth muscle cells is increased by the accumulation of intracellular Na+ and subsequent elevation of cytoplasmic Ca++ concentration. In contrast to the hemodynamic effects of osmotically active Na+, osmotically inactive Na+ stimulates immune cells and produces proinflammatory cytokines, which contribute to hypertension. When this occurs in the gut, the microbiota may become imbalanced, leading to intestinal inflammation and systemic hypertension. In conclusion, the primary cause of hypertension is sodium overload resulting from kidney dysregulation.

Accumulation patterns of intracellular salts in a new halophilic amoeboflagellate, Euplaesiobystra salpumilio sp. nov., (Heterolobosea; Discoba) under hypersaline conditions

Halophilic microbial eukaryotes are present in many eukaryotic lineages and major groups; however, our knowledge of their diversity is still limited. Furthermore, almost nothing is known about the intracellular accumulation of salts in most halophilic eukaryotes. Here, we isolate a novel halophilic microbial eukaryote from hypersaline water of 134 practical salinity units (PSU) in a solar saltern. This species is an amoeboflagellate (capable of the amoeba-flagellate-cyst transformation) in the heterolobosean group and belongs to the genus Euplaesiobystra based on morphological data and 18S rDNA sequences. However, the isolate is distinct from any of the described Euplaesiobystra species. Especially, it is the smallest Euplaesiobystra to date, has a distinct cytostome, and grows optimally at 75–100 PSU. Furthermore, the phylogenetic tree of the 18S rDNA sequences demonstrates that the isolate forms a strongly supported group, sister to Euplaesiobystra hypersalinica. Thus, we propose that the isolate, Euplaesiobystra salpumilio, is a novel species. E. salpumilio displays a significantly increased influx of the intracellular Na+ and K+ at 50, 100, and 150 PSU, compared to freshwater species. However, the intracellular retention of the Na+ and K+ at 150 PSU does not significantly differ from 100 PSU, suggesting that E. salpumilio can extrude the Na+ and K+ from cells under high-salinity conditions. Interestingly, actively growing E. salpumilio at 100 and 150 PSU may require more intracellular accumulation of Na+ than the no-growth but-viable state at 50 PSU. It seems that our isolate displays two salt metabolisms depending on the tested salinities. E. salpumilio shows a salt-in strategy for Na+ at lower salinity of 100 PSU, while it displays a salt-out strategy for Na+ at higher salinity of 150 PSU. Our results suggest that the novel halophilic E. salpumilio fundamentally uses a salt-out strategy at higher salinities, and the accumulation patterns of intracellular salts in this species are different from those in other halophilic microbial eukaryotes.

Increased susceptibility to ventricular arrhythmia at low-normal and moderately low levels of extracellular potassium in catecholaminergic polymorphic ventricular tachycardia.

Recent studies have shown that the predominant arrhythmogenic mechanism in hypokalemia is through inhibition of cardiac Na+/K+ ATPase [Figure 1A(1)]. This results in accumulation of intracellular Na+, which reduces the concentration gradient across the sodium/calcium exchanger [Figure 1A(2)]. The resultant Ca2+ buildup activates CaMKII, an enzyme that facilitates further Na+ and Ca2+ accumulation by activating cardiac ryanodine receptors (RyR2), L-type Ca2+ channels, phospholamban, and the late sodium current [Figure 1A(3)].

Slow [Na+]i dynamics impacts arrhythmogenesis and spiral wave reentry in cardiac myocyte ionic model.

Accumulation of intracellular Na+ is gaining recognition as an important regulator of cardiac myocyte electrophysiology. The intracellular Na+ concentration can be an important determinant of the cardiac action potential duration, can modulate the tissue-level conduction of excitation waves, and can alter vulnerability to arrhythmias. Mathematical models of cardiac electrophysiology often incorporate a dynamic intracellular Na+ concentration, which changes much more slowly than the remaining variables. We investigated the dependence of several arrhythmogenesis-related factors on [Na+]i in a mathematical model of the human atrial action potential. In cell simulations, we found that [Na+]i accumulation stabilizes the action potential duration to variations in several conductances and that the slow dynamics of [Na+]i impacts bifurcations to pro-arrhythmic afterdepolarizations, causing intermittency between different rhythms. In long-lasting tissue simulations of spiral wave reentry, [Na+]i becomes spatially heterogeneous with a decreased area around the spiral wave rotation center. This heterogeneous region forms a functional anchor, resulting in diminished meandering of the spiral wave. Our findings suggest that slow, physiological, rate-dependent variations in [Na+]i may play complex roles in cellular and tissue-level cardiac dynamics.

Live fluorescence and transmission-through-dye microscopic study of actinomycin D-induced apoptosis and apoptotic volume decrease

The effect of actinomycin D on HeLa cells was studied by live fluorescence and transmission-through-dye microscopy-a recently developed technique that permits volume measurements in live cells. In particular, it is well suited for the observation and quantification of the apoptotic volume decrease (AVD), which is widely viewed as an essential feature of apoptosis. The main results from our study are as follows. (1) Apoptosis caused in HeLa cells by actinomycin D proceeds in two morphologically distinct stages: the early stage is characterized by extensive blebbing, and the late stage by a more compact shape. The loss of mitochondrial membrane potential occurs at about the same time as blebbing, and chromatin condensation follows 30-90 min later. Caspase-3 and 7 become activated during the late stage. (2) Because blebbing occurs before activation of caspase-3, it has to be initiated by a different mechanism. Although blebbing is one of the earliest observable changes, it can be selectively inhibited without affecting other apoptotic reactions. (3) The majority of cells experience a temporary volume increase after the appearance of blebs. Eventually, AVD takes over and the cells shrink by approximately 40 % of their initial volume; the volume loss becomes noticeable at the end of the blebbing phase and continues through the late stage. Sometimes, at the end of long incubations, shrinkage gives way to swelling, possibly indicating secondary necrosis. (4) Both early and late apoptosis are accompanied by intracellular accumulation of Na(+), while low-sodium medium prevents apoptosis. Except for a partial protective effect of quinine, all of the tested blockers of Na(+), K(+) and Cl(-) channels failed to prevent apoptosis or AVD.

Influence of intracellular Na+, K+ and Cl- on the salt tolerance in suspension cell cultures of Medicago media

In the process of selection for salt tolerance, it is important to understand the physiological basis of ion management executed by the cells through the exclusion, accumulation or maintenance of ratios of specific ions. Intracellular accumulation of Na + , K + and Cl- ions in the cells in vitro was studied as a factor in salt tolerance in suspension cultures of Medicago media cv. Rambler. Cells selected for NaCl tolerance (R151-S) and the non-selected R151 (normal) cells were grown in MS medium containing 0, 100, 250, 400 and 550 mM NaCl, Na + , Cl - or K + for 5 weeks and examined for relative growth rate per week (RGR/week) and intracellular concentration of Na + , Cl - and K + ions. R151-S cells were found to be significantly more tolerant to the elevated Na + and Cl- levels than R151. When the effects of Na + and Cl - ions were compared, R151-S appeared to be more tolerant of higher concentrations of Cl - as compared to the equimolar concentrations of Na + . As the concentration of NaCl in the medium increased, the intracellular concentrations of Na + increased, though not at a proportional rate. Also, when the external Cl- concentration increased, there was no significant change in the intracellular accumulation of Na + . On the contrary, intracellular Na + decreased when the external K + was increased. In the cells adapted to high concentration of NaCl, the concentration of intracellular K + was lower than the non-adapted cells. The adequate intracellular K + concentration for optimum growth in M. media suspension cultures was found to be 3000 µM/g dry weight. Excess or deficiency of intracellular K + caused severe reduction in the growth rates in all cell lines. Key words : Salt tolerance, Medicago media, suspension cell culture, intracellular ions, organic relative growth rate.

Amyotrophic lateral sclerosis and the neuroprotective potential of exercise

When confronted with the devastating diagnosis of amyotrophic lateral sclerosis (ALS, or motor neurone disease), patients are desperate for information particularly in regards to causation and treatment. Inevitably, discussion turns to lifestyle-related issues, and often the role of exercise. Unfortunately, this is a complex, largely unresolved area of disease management, with the potential benefits of exercise in ALS remaining an open discussion between physician and patient. In America, ALS is also known as Lou Gehrig's disease, named after the famous New York Yankees baseballer (Fig. 1). Gehrig was a superb athlete, and as with most ALS patients, there seemed to be no apparent reason for him to contract this universally fatal disease at the young age of 35, and die 2 years later. Other seemingly random cases have subsequently been described in high-performance athletes. However, a recent retrospective study of more that 7000 footballers from the Italian professional leagues established that standardized morbidity ratios were increased for the development of ALS, particularly younger onset (Chioet al. 2005). For unexplained reasons, footballers who played for more than 5 years, particularly in an active midfield position, were at highest risk. Other studies have suggested a raised risk of ALS in marathon runners, and further epidemiological investigations are currently underway in rugby populations. Figure 1 Lou Gehrig, the New York Yankees baseballer, who developed ALS in his mid-thirties. Although ALS is typically sporadic, approximately 10% of cases are familial, whereby two or more family members are clinically affected. The exact pathophysiological mechanisms underlying neurodegeneration in both familial and sporadic ALS have yet to be defined. Inheritance of familial ALS is usually autosomal dominant, often linked to mutation of the copper/zinc superoxide-dismutase-1 gene (SOD-1) resulting in the typical adult-onset ALS phenotype (for review, see Vucic & Kiernan, 2009). The key function of the SOD-1 enzyme involves free radical scavenging, with the enzyme catalysing the conversion of the superoxide anion to molecular oxygen and hydrogen peroxide. The processes underlying ALS appear multifactorial, involving complex interaction between genetic factors and molecular pathways, with resultant damage of critical target proteins and organelles within the motor neurone. In terms of any pathophysiological link between exercise and the development of ALS, oxidative stress, excessive free radical production and increased glutamate stimulation may all accompany normal motor neurone activation. However, it is plausible that such processes may potentially become neurotoxic as a result of exercise and excessive activition in susceptible individuals. Dysfunction of the energy-dependent, axonal Na+/K+ electrogenic pump may also be an integral factor contributing to motor neurone loss in ALS (Vucic et al. 2007). In addition to regulating the resting membrane potential, the Na+/K+ pump exerts a significant role in the restoration of Na+ and K+ gradients after high-frequency impulse activity. Specifically, after a period of prolonged activity, the accumulated intracellular Na+ ions increase Na+/K+ pump activity resulting in membrane hyperpolarization. In the setting of a dysfunctional Na+/K+ pump, exercise may conceivably result in accumulation of intracellular Na+ and thereby provoke disruption of resting membrane potential. Secondary effects mediated by changes in intracellular Na+, including reverse activation of the Na+–Ca2+ exchanger, would lead to intracellular increases in Ca2+ concentration, activation of calcium-dependent enzyme systems and neuronal death. Of relevance, widespread loss and dysfunction of Na+/K+ pump function have been demonstrated in the SOD-1 ALS mouse model. Such discussion remains largely hypothetical and relates more to a potential role for exercise in the causation of ALS. How then, should the treating neurologist advise patients once diagnosed with ALS regarding their lifestyle and specifically, the role of exercise? Unfortunately a recent Cochrane analysis cited a lack of randomised clinical trial data that may guide appropriate discussion concerning the potential benefits or risks of aerobic type exercise in ALS (Dalbello-Haas et al. 2008). Similarly, there is limited information concerning resistance training, although recent pilot studies involving inspiratory muscle training suggest potential benefit, with larger multicentre studies currently underway (Cheah et al. 2009). Given a lack of clinical trial information, the study by Deforges and colleagues in this issue of The Journal of Physiology is timely and potentially encouraging (Deforges et al. 2009). Specifically, using a targeted programme involving swimming, the authors demonstrated improved motor function, protection of motor neurones and their supportive cell structures, and ultimately increased lifespan in the G93A transgenic SOD-1 mouse model of ALS. These findings in a SOD-1 mouse model of ALS must be interpreted with caution, particularly given the fact that of the many (>100) trials undertaken in SOD-1 mouse models of ALS, none of the positive findings from animal studies have translated into meaningful therapy in human ALS. As it stands, the recent findings related to exercise clearly need further validation, but perhaps they may serve to increase the momentum for larger scale, exercise-based clinical trials in ALS patients.

Influences of nanoparticle zinc oxide on acutely isolated rat hippocampal CA3 pyramidal neurons

The effects of zinc oxide nanoparticles (nano-ZnO) on the properties of voltage-dependent sodium, potassium currents and evoked action potentials were studied in acutely isolated rat hippocampal CA3 pyramidal neurons at postnatal ages of 10–14 days rats using the whole-cell patch-clamp technique. The results indicated that: (1) in the present of final concentration of 10 −4 g/ml nano-ZnO, the current–voltage curve of sodium current ( I Na) was decreased, and the peak amplitudes of I Na were increased considerably from −50 to +20 mV ( p < 0.05). Meanwhile, the inactivation and the recovery from inactivation of I Na were also promoted by the nano-ZnO solution (10 −4 g/ml) ( p < 0.01). However, the steady-state activation curve of I Na was not shifted by the nano-ZnO. (2) The amplitudes of transient outward potassium current ( I A) were increased by the nano-ZnO solution (10 −4 g/ml), while the current–voltage curve of delayed rectifier potassium current ( I K) was significantly increased from +20 to +90 mV ( p < 0.05). However, it is apparent that the nano-ZnO solution did not shift the steady-state activation curve of I A and I K, and neither had significant effects on the inactivation and the recovery from inactivation of I A. (3) Peak amplitude and overshoot of the evoked single action potential were increased and half-width was diminished in the presence of the 10 −4 g/ml nano-ZnO solution ( p < 0.05). Simultaneously, a prolonged depolarizing current injection enhanced ( p < 0.05) repetitive firing evoked firing rate. These results suggested that 10 −4 g/ml nano-ZnO solution can lead to an enhancement in the current amplitudes of I Na and I K by increasing the opening number of sodium channels, delaying rectifier potassium channels, and enhancing the excitability of neurons, which lead to Na + influx and the accumulation of intracellular Na +, as well as K + efflux plus the loss of cytoplasmic K +. These may disturb the ionic homeostasis and the physiological functions of neurons.

Mitochondrial Dysfunction and Dendritic Beading during Neuronal Toxicity

Mitochondrial dysfunction (depolarization and structural collapse), cytosolic ATP depletion, and neuritic beading are early hallmarks of neuronal toxicity induced in a variety of pathological conditions. We show that, following global exposure to glutamate, mitochondrial changes are spatially and temporally coincident with dendritic bead formation. During oxygen-glucose deprivation, mitochondrial depolarization precedes mitochondrial collapse, which in turn is followed by dendritic beading. These events travel as a wave of activity from distal dendrites toward the neuronal cell body. Despite the spatiotemporal relationship between dysfunctional mitochondria and dendritic beads, mitochondrial depolarization and cytoplasmic ATP depletion do not trigger these events. However, mitochondrial dysfunction increases neuronal vulnerability to these morphological changes during normal physiological activity. Our findings support a mechanism whereby, during glutamate excitotoxicity, Ca(2+) influx leads to mitochondrial depolarization, whereas Na(+) influx leads to an unsustainable increase in ATP demand (Na(+),K(+)-ATPase activity). This leads to a drop in ATP levels, an accumulation of intracellular Na(+) ions, and the subsequent influx of water, leading to microtubule depolymerization, mitochondrial collapse, and dendritic beading. Following the removal of a glutamate challenge, dendritic recovery is dependent upon the integrity of the mitochondrial membrane potential, but not on a resumption of ATP synthesis or Na(+),K(+)-ATPase activity. Thus, dendritic recovery is not a passive reversal of the events that induce dendritic beading. These findings suggest that the degree of calcium influx and mitochondrial depolarization inflicted by a neurotoxic challenge, determines the ability of the neuron to recover its normal morphology.

Contribution of Protons to Post‐Ischemic Na+ and Ca2+ Overload and Left Ventricular Mechanical Dysfunction

The intracellular accumulation of Na(+) and Ca(2+) plays a key role in ischemia-induced myocardial injury that may be manifest as left ventricular (LV) mechanical dysfunction, dysrhythmias, or infarction. This review considers the potential contributions of protons (H(+)) produced during ischemia as well as reperfusion to intracellular Na(+) and Ca(2+) homeostasis. ATP hydrolysis produces H(+) and the resulting intracellular acidosis directly impairs LV contractility. However, it is the accumulation of intracellular H(+) and the activation of Na(+)-dependent pH regulatory mechanisms, including the Na(+)-H(+) exchanger (NHE-1) and the Na(+)-HCO(3) (-) cotransporter, which contribute to Na(+) accumulation. Intracellular Na(+) accumulation, coupled with the NHE-1, then causes Ca(2+) overload and further LV mechanical dysfunction. As glycolysis uncoupled from glucose oxidation is an important determinant of the rate of H(+) production, factors that affect glucose metabolism, including degree of ischemia, myocardial workload, and competition from other energy substrates, are expected to influence Na(+) and Ca(2+) accumulation, and hence the recovery of post-ischemic LV mechanical function. Whereas an increase in the uncoupling of glycolysis from glucose oxidation accelerates H(+) production and worsens the recovery of LV mechanical function, inhibition of H(+) production improves recovery of post-ischemic LV mechanical function. Thus, alteration of glucose metabolism, either by inhibition of an excessive rate of glycolysis or by stimulation of glucose oxidation, is an attractive drug target to reduce H(+) production and limit Na(+) and Ca(2+) accumulation and thereby prevent post-ischemic LV dysfunction.

A novel toxicity-based assay for the identification of modulators of voltage-gated Na + channels

Voltage-gated Na + channels are promising drug targets. Screening of large numbers of putative modulators, however, can be demanding and expensive. In this study, a simple, cheap, and robust assay to test the pharmacological modulation of Na + channel function is presented. The assay makes use of the fact that the intracellular accumulation of Na + ions can be cytotoxic. The toxicity of the Na + channel activator veratridine in the presence of an inhibitor of the Na +/K +ATPase (ouabain) in a Nav1.2a (rat brain IIA α) expressing cell line is assessed. Na + channel blockers should reduce toxicity in this model. CHO cells which recombinantly expressed rat Nav1.2a subunits were seeded in 96-well plates, and cell survival was tested after 24 h incubation in medium containing veratridine and ouabain in the presence or absence of Na + channel blockers. Propidium iodide fluorescence was used as toxicity readout. Veratridine (100 μM) or ouabain alone (500 μM) were not toxic to the cells. In the presence of 500 μM ouabain, however, veratridine induced halfmaximal cell death with an EC 50 value of 15.1±2.3 μM. Ouabain’s EC 50 was 215.3±16.7 μM (with 30 μM veratridine). The effects of a number of Na + channel blockers were tested and compared with their Na + channel blocking activity measured in voltage-clamp experiments. Blockers from various chemical classes reduced toxicity half maximally with IC 50 values ranging from 11.7±1.4 nM (tetrodotoxin) to 280.5±48.0 μM (lamotrigine). There was a linear relationship between the log IC 50 values obtained by the two methods (slope: 1.1±0.08; correlation coefficient: 0.93). In summary, these data show that this novel toxicity assay is well suited to test Na + channel blockers.

Na-K-Cl Cotransporter Contributes to Glutamate-Mediated Excitotoxicity

We hypothesized that cation-dependent Cl- transport protein Na-K-Cl cotransporter isoform 1 (NKCC1) plays a role in the disruption of ion homeostasis in cerebral ischemia. In the current study, a role for NKCC1 in neuronal death was elucidated in neurotoxicity induced by glutamate and oxygen and glucose deprivation (OGD). Incubation of cortical neurons cultured for 14-15 d in vitro (DIV) with 100 microm glutamate for 24 hr resulted in 50% cell death. Three hours of OGD followed by 21 hr of reoxygenation led to 70% cell death. Inhibition of NMDA receptors with dizocilpine hydrogen maleate (1 microm) prevented both OGD- and glutamate-mediated cell death. Moreover, blocking of NKCC1 activity with bumetanide (5-10 microm) abolished glutamate- or OGD-induced neurotoxicity. Bumetanide was ineffective if added after 10-120 min of glutamate incubation or 3-6 hr of OGD treatment. Accumulation of intracellular Na+ and 36Cl content after NMDA receptor activation was inhibited by bumetanide. Blockage of NKCC1 significantly attenuated cell swelling after OGD or NMDA receptor activation. This neuroprotection was age dependent. Inhibition of NKCC1 did not protect DIV 7-8 neurons against OGD-mediated cell death. In contrast, cell death in DIV 7-8 neurons was prevented by the protein-synthesis inhibitor, cycloheximide. Taken together, the results suggest that NKCC1 activity is involved in the acute excitotoxicity as a result of excessive Na+ and Cl- entry and disruption of ion homeostasis.

Involvement of intracellular Na+accumulation in Hg+2or Cd+2induced cytotoxicity

Previously we showed that hepatocyte lysis induced by Hg +2 or Cd +2 could be partly attributed to mitochondrial toxicity [1,2]. Similar changes in Na + homeostasis induced when Cd +2 or Hg +2 was incubated with hepatocytes. Cd +2 or Hg +2 induced cytotoxicity were prevented by Na + omission from the media or by the addition of the Na + / H + exchange inhibitor 5-(N,N-dimethyl)-amiloride. Furthermore the omission of Cl - from the media or addition of glycine, a Cl - channel blocker also prevented Cd +2 or Hg +2 induced hepatocyte toxicity. A hypotonic media also increased Cd +2 or Hg +2 induced hepatocyte cytotoxicity. This suggests that Cd +2 or Hg +2 cytotoxicity could be partly attributed to disruption of cell volume regulation mechanisms. The increased osmotic load caused by the uncontrolled accumulation of intracellular Na + in Cd +2 or Hg +2 treated hepatocytes likely resulted from the activation of Na + / H + exchanger and the Na + / HCO3 - cotransporter by the acidosis and ATP depletion caused by mitochondrial toxicity.

Possible Role of a Neuropeptide PACAP (Pituitary Adenylate Cyclase‐Activating Polypeptide) on Stimulus‐Secretion Coupling in Catecholamine Neuron

AbstractFor Abstract see ChemInform Abstract in Full Text.

Ouabain changes arterial blood pressure and vascular reactivity to phenylephrine in L-NAME-induced hypertension.

Ouabain is an endogenous compound that has been associated with the genesis and maintenance of hypertension. This compound inhibits the Na+ pump activity, which leads to an accumulation of intracellular Na and ultimately might increase vascular tone. In nanomolar concentrations, it enhances vasopressor responses to phenylephrine in some vascular beds from normotensive and hypertensive rats. However, it is not known whether this action of ouabain is a common mechanism for all models of hypertension. The aim of this work was to determine whether ouabain can alter pressor responses to phenylephrine in rats with Nomega-nitro-l-arginine methyl ester (L-NAME)-induced hypertension. In anesthetized rats, ouabain (0.18 microg/kg, i.v.) increased arterial blood pressure in L-NAME-treated rats but not in controls. Ganglionic blockade by hexamethonium (5 mg/kg, i.v.) prevented the increase in arterial blood pressure produced by ouabain in L-NAME-treated rats. Additional studies using isolated perfused tail artery preparations were performed to investigate which factors are involved in the action of ouabain in L-NAME-treated rats. The effects of 10 nM ouabain on the vasoconstrictor actions of phenylephrine were determined on preparations with intact or damaged endothelium or in the presence of tetraethylammonium (a K+-channel blocker). Ouabain reduced pressor actions of phenylephrine in preparations with an intact endothelium. However, after endothelial damage or infusing tetraethylammonium, the response to phenylephrine was increased after ouabain. In tails from L-NAME-treated rats, the functional activity of the Na, K+-ATPase was reduced, and 10 nM ouabain did not produce any further reduction. In conclusion, in this model of hypertension, a low dose of ouabain (0.18 microg/kg) increased arterial blood pressure in vivo probably as a result of increased sympathetic tone. However, this effect was not accompanied by an enhanced action of phenylephrine on the tail vascular bed with an intact endothelium. The results suggest that this was due to the release of an endothelium-derived K+-channel opener.

Sensitivity of oocyte-expressed epithelial Na+ channel to glibenclamide

The effect of glibenclamide on heterologously expressed amiloride-sensitive sodium channels (ENaCs) was investigated in Xenopus oocytes. The ENaC is a heteromer and consists of α-, β- and γ-subunits and the α- and β-subunits have previously been shown to confer sensitivity to glibenclamide. We coexpressed either colonic rat α- (rα) or guinea-pig α-subunit (gpα) with Xenopus βγ-subunits. The gpαxβγ was significantly stimulated by glibenclamide (100 μM) (184±15%), whereas the rα-combination was slightly down-regulated by the sulfonylurea (79±4%). The stimulating effect did not interfere with Na+-self-inhibition resulting from intracellular accumulation of Na+-ions. We exchanged cytosolic termini between both orthologs but the gpα-chimera with the termini from rat retained sensitivity to glibenclamide. The effect of glibenclamide on Xenopus ENaC (xENaC) was inhibited by ADP-β-S but not by ATP-γ-S, when applied intracellularly. Intracellular loading with Na+-ions after inhibition of Na+/K+-ATPases with ouabain prevented an up-regulation of ENaC activity by glibenclamide. Pretreatment of oocytes expressing xENaC with edelfosine (ET-18-OCH3) slightly reduced stimulation of Iami (118±12%; control: 132±9%) while phosphatidylinositol-4,5-biphosphate (PIP2) significantly reduced the effect of glibenclamide to 101±3%.

Possible role of a neuropeptide PACAP (pituitary adenylate cyclase-activating polypeptide) on stimulus-secretion coupling in catecholamine neuron

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide first isolated from ovine hypothalamic tissue. This peptide stimulates adenylate cyclase activation. However, few details were known of the function of this peptide on stimulus-secretion coupling in neuronal cells. The authors have investigated the role of PACAP on catecholamine biosynthesis and secretion using cultured bovine adrenal chromaffin cells as a model for catecholamine-containing neurons. PACAP38, the 38-amino acid form of PACAP, increased cAMP formation in bovine adrenal chromaffin cells. In addition, PACAP38 increased [Ca2+]i associated with PI turnover and Ca2+ influx into the cells. The synthesis of catecholamine and the phosphorylation of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis, stimulated by the maximal effective concentration of dibutyryl cAMP or a high concentration (56 mM) of K+ were further enhanced by PACAP38. Thus PACAP38 stimulated the pathway of catecholamine biosynthesis mainly by both activation of cAMP- and Ca2(+)-dependent protein kinases. On catecholamine secretion from the cells, the effect of PACAP38 was markedly potentiated by addition of ouabain, an inhibitor of Na+/K+ ATPase. This markedly potentiated secretion was greatly reduced with Na+ omitted-sucrose medium. PACAP38 increased 22Na+ influx into the cells treated with ouabain. Thus PACAP38 with ouabain stimulated catecholamine secretion by accumulation of intracellular Na+, resulting in an increase in Ca2+ influx. These results indicate that the neuropeptide PACAP has an important role in stimulus-secretion coupling in adrenal chromaffin cells.

Role of voltage-gated Na+ channels in hypoxia-induced neuronal injuries.

1. Mammalian neurons in the central nervous system are vulnerable to oxygen deprivation. In clinical conditions, such as stroke or apnoea, permanent loss of neuronal functions can occur within minutes of severe hypoxia. 2. Recent studies have focused on the role of Na+ in acute neuronal responses to hypoxia. These studies have shown that the influx of extracellular Na+ is an important factor in hypoxia-induced injury and that blockade of voltage-gated Na+ channels reduces hypoxic responses and injury of neurons. Yet, the mechanism underlying the effect of blockade of Na+ channels on hypoxic injury is unclear. 3. The aim of the present review is to discuss the above topics given the current understanding of the role of Na+ channels in hypoxia and its implications on therapeutic strategy for preventing hypoxia-induced neurological damage. 4. It has been known that the maintenance of ionic homeostasis and membrane properties in neurons are improved by reducing the activity of voltaged-gated Na+ channels during acute hypoxia. 5. Recent studies suggest that persistent Na+ current and Na+-dependent exchangers may play a role in Na+ influx and neuronal injury during hypoxia. 6. The neuroprotective action of blockers of the Na+ channel may also be via the improved maintainance of intracellular energy levels because the action is dependent on cellular energy levels and extracellular glucose during hypoxia. 7. Hence, the blockade of voltage-gated Na+ channels reduces the excitability of neurons, Na+ influx and the accumulation of intracellular Na+. These improve the ionic homeostasis and cellular energy levels and, thus, prevent hypoxia-induced neuronal injury and neuronal damage mediated by Ca2+ overload.

Mechanisms of arachidonic acid induced glial swelling

Accumulation of arachidonic acid (AA) in the brain during ischaemia may contribute to development of brain oedema. In this study we investigated the effect of selected drugs on AA-induced cytotoxic brain oedema in C6 glioma cells. Suspended C6 glioma cells were preincubated with drugs and AA (0.1 mM) was added. When no drug was administered cell volume increased immediately after the addition of AA with a maximum cell swelling of 13.1±1.9% at 15 min (mean±S.E.M.). Preincubation of cells with BW 755C, a dual inhibitor of cyclo- and lipoxygenases, showed no reduction in cell swelling from AA, whereas superoxide dismutase, amiloride and the protein kinase inhibitor H-9370 led to a significant attenuation of volume increase ( p<0.05). The role of Na + ions during cell swelling from AA was evaluated after pretreatment of C6 glioma cells with ouabain. This resulted in a reversal of cell swelling ( p<0.01). We conclude that there is potential involvement of free radicals, signal transduction systems and intracellular accumulation of Na + ions in glial cell swelling from AA.

Effect of hypothermia on the volume of rat glial cells.

1. The cell volume of suspended C6 glioma cells and primary cultured rat astrocytes was measured at normothermia (37 degrees C), and at mild (32 degrees C) and moderate (27 degrees C) hypothermia by flow cytometry with electrical cell sizing. 2. Under control conditions (37 degrees C), C6 glioma cells had a volume of 809 +/- 29 microm3. Moderate hypothermia (27 degrees C) led to rapid cell swelling, with a maximum volume of 113.1 +/- 1.3 % of control being achieved after 50 min. After rewarming to 37 degrees C, cell volume recovered very slowly and incompletely (to 107.2 +/- 0.4 % of control). Less severe hypothermia (32 degrees C) led to a smaller increase in cell volume (108.7 +/- 0.5 % of control). 3. The maximal cell swelling response and the kinetics of swelling were similar in C6 glioma cells and primary cultured astrocytes. 4. Hypothermia-induced cell swelling was dependent on the presence of extracellular Na+ and was reduced by the Na+-H+ antiporter inhibitor EIPA. 5. The underlying mechanisms of hypothermia-induced cell swelling are an intracellular accumulation of Na+ by (1) differential effects of hypothermia on the membrane permeabilities of Na+ and K+ and (2) activation of the Na+-H+ antiporter by a shift of its activation curve to a more alkaline value.