Intracellular Sodium Concentration Research Articles

Neuroprotective effect of fish oil in diabetic neuropathy.

Diabetic neuropathy is a degenerative complication of diabetes accompanied by an alteration of nerve conduction velocity and Na,K-ATPase activity. This decrease in Na,KATPase activity has been implicated in the pathogenesis of electrophysiological abnormalities characterizing neuropathic complication in different animal models and in humans (1). Na,K-ATPase is a membrane-bound enzyme consisting of three catalytic isoenzymes (~1, ct2, ~3) and a large lipid core (2). It plays a fundamental rote in Na + and K + transport by maintaining the ionic gradients necessary for nervous excitability. A decrease in this process leads to an increase in the intracellular concentration of sodium and a corresponding decrease in membrane sodium potential. The mechanism responsible for impairment of Na,K-ATPase during hyperglycemia is still uncleal, but alterations of the kinetic parameters of the enzyme and/or of subunit expression are likely (3,4). In recent years there has been great interest in the effect of n-3 essential fatty acids, found particularly in fish oil, on the prevention of atherosclerosis and hypertension in animal models and patients with vascular disease (5). Fish oil treatment as MaxEPA was recently reported to improve vascular function in diabetic patients (6), and prevent c~2 isoenzyme activity of Na, K-ATPase in diabetic cardiomyopathy (7). Aim o f the study. The present study in rats was designed, first, to measure diabetes-induced abnormalities in Na,KATPase acyivity, isoenzyme expression, fatty acid content in sciatic membranes, and nerve conduction velocity and second, to assess the preventive ability of a fish-oil rich diet (rich in n-3 fatty acids) on these abnormalities. Methods. Diabetes was induced by intravenous streptozotocin injection. Diabetic animals (D) and nondiabetic control animals (C) were fed the standard rat chow either without supplementation or supplemented with either fish oil (DM, CM) or olive oil (DO, CO) at a daily dose of 0.5 g/kg by gavage during 8 wk.

Epidermal growth factor accelerates recovery of LLC-PK1 cells following oxidant injury.

In renal tubular epithelial cells, oxidant injury results in several metabolic alterations including ATP depletion, decreased Na+K+ATPase activity, and altered intracellular sodium and potassium content. To investigate the recovery of LLC-PK1 cells following oxidant injury and to determine if recovery can be accelerated, we induced oxidant stress in LLC-PK1 cells with 500 microM hydrogen peroxide for 60 min. Identical cohorts of oxidant-stressed cells were incubated in recovery medium without epidermal growth factor (EGF) or recovery medium containing 25 ng EGF per ml. ATP levels, Na+K+ATPase activity in whole cells, Na+K+ATPase activity in disrupted cells, and intracellular sodium and potassium ion content were determined at 0, 5, 24, 48, and 72 h following oxidant injury in each cohort of cells. In oxidant-stressed cells recovering in medium without EGF, ATP levels, Na+K+ATPase activity, and intracellular ion content improved but continued to remain substantially lower than control values at all time points following oxidant stress. In cells recovering in medium with EGF, ATP levels, Na+K+ATPase activity, and the intracellular potassium-to-sodium ratio were significantly higher at nearly all time points than values in cells recovering in medium alone. In cells recovering with added EGF, Na+K+ATPase activity had improved to control levels, whereas ATP levels and intracellular ion content approached control values by 72 h following oxidant stress. We conclude that oxidant-mediated ATP depletion, altered Na+K+ATPase activity, and intracellular ion content remain depressed for several d following oxidant stress and that EGF accelerated recovery of LLC-PK1 cells from oxidant injury.

Early sodium elevations induced by combined oxygen and glucose deprivation in pyramidal cortical neurons.

We investigated the effects of oxygen (O2)/glucose deprivation on intracellular sodium concentration ([Na+]i) of cortical pyramidal cells in a slice preparation of rat frontal cortex. Intracellular recordings were combined with microfluorometric measurements of [Na+]i using the Na+-sensitive dye sodium-binding benzofuran isophthalate (SBFI). Deprivation of O2/glucose caused an initial membrane hyperpolarization that was followed by a slowly developing large depolarization. Levels of [Na+]i started to increase significantly during the phase of membrane hyperpolarization. Neither tetrodotoxin, a combination of ionotropic and metabotropic glutamate receptor antagonists (D-amino-phosphonovalerate, 6-cyano-7-nitroquinoxaline-2,3-dione plus S-methyl-4-carboxyphenylglycine) nor bepridil, an inhibitor of the Na+/Ca2+-exchanger, affected these responses to O2/ glucose. The present results demonstrate that, in cortical neurons, O2/glucose deprivation induces an early rise in [Na+]i which cannot be ascribed to the activity of voltage gated Na+-channels, glutamate receptors or of the Na+/Ca2+-exchanger.

Increased Na+ and decreased Mg2+ intracellular concentrations in vascular smooth muscle cells from spontaneously hypertensive rats.

1. Although in blood cells decreased magnesium concentrations and increased sodium concentrations in essential hypertension have often been described, only sparse data exist on cellular magnesium or sodium content and exchange in vascular smooth muscle cells.2. Therefore in aortic smooth muscle cells from 10 spontaneously hypertensive rats (SHR) of the Münster strain and 10 normotensive Wistar-Kyoto rats (WKY) aged 3 and 8-10 months, the intracellular magnesium and sodium content was measured.3.Electron-probe X-ray microanalysis was used to determine intracellular Mg2+ and Na+ concentrations in aortic cryosections 3 micron thick. The Mg2+ content was 47+/-13 mmol/kg dry weight in SHR versus 48+/-19 mmol/kg dry weight in WKY aged 3 months, and 37+/-6 mmol/kg dry weight in SHR versus 47+/-4 mmol/kg dry weight in WKY aged 8-10 months (P<0. 05). Vascular smooth muscle Na+ content was 283+/-59 mmol/kg dry weight in WKY and 402+/-123 mmol/kg dry weight in SHR aged 3 months (P<0.05), and 289+/-17 mmol/kg dry weight in WKY versus 548+/-39 mmol/kg dry weight in SHR aged 8-10 months (P<0.05).4. Aortic smooth muscle cells from SHR are characterized by a markedly lower intracellular Mg2+ content in 8-10-month-old animals and increased Na+ concentrations compared with normotensive cells in 3- and 8-10-month-old rats. The results may be due to genetically determined disturbances in transmembrane Mg2+ and Na+ transport. Cellular magnesium and sodium handling may be disturbed in SHR aortic smooth muscle as it is in hypertensive blood cells. In addition, it is concluded that vascular smooth muscle cell Mg2+-Na+ exchanger can be altered in a subgroup of SHR.

Fluorometric analysis of intracellular sodium concentrations in isolated retinal horizontal cells.

under these conditions was lowered to approximately 10 /.LV, supporting the contention that the differential voltage being measured by the electrode results from hydrogen ions. A background recording was then again made 160 pm away from the cell. Finally, the electrode was returned to the cell and the dish refreshed with 2 mM HEPES Ringer, resulting in restoration of the signal. Our results demonstrate that self-referencing pH-selective microelectrodes can indeed be used to examine H’ net flux across the membranes of isolated retinal neurons. We are now employing specific pharmacological probes that selectively inhibit various pH transport mechanisms in an effort to clarify the mechanisms responsible for the net fluxes we have detected. We are grateful to Richard H. Sanger for electronic and computer assistance and Rudi Rottenfusser of the Zeiss Corporation for the generous loan of microscope equipment. This work was supported by grants EYO 9411 from the National Eye Institute, P41 RR01 395 from the National Center for Research Resources, and grant DBI-9605155 from the National Science Foundation. 1. 2. 3.

A New EDS Equipment (GEM Detector With Superatw Window) and a Correction for Si-Contamination Improve the Quantitative Electron Probe Micro Analysis of Biological Cryosections

Abstract Function dependent changes in the intracellular (i.c.) concentrations of calcium and sodium play a crucial role in the regulation of biological processes such as contraction of heart muscle. However, accurate measurements of both elements are difficult since in i.e. compartments they are present in low concentrations, hence the Ca and Na peaks show small count rates and small peak to continuum ratios. The accurate quantification of Ca and Na is rendered even more difficult by the partially overlap of the NaK with CuL peak and CaKα with KKβ peak. In some cases electrophysiologist substitute in the experiments K+ with Cs+ or the pathologist is interested in the storage of mercury. For analysis of such elements (Cs, Hg) with higher atomic number the stopping power of the EDS crystal play an important role for system sensitivity. Quantitative analysis of thin biological cryosections with the continuum method must be corrected for influence of X-rays emit from holder, supporting film and grid.

EDS of thin Biological Specimen in the Study of Time-Dependent Physiological Processes

Abstract The contraction of muscle depends on the ability of intracellular organelles to rapidly release and take up large amounts of activator calcium. In cardiac muscle the intracellular sodium concentration is known to also modulate this Ca2+ release: Na+-Ca2+ exchange creates narrow cytosolic microdomains just internal to the sarcolemma (approximately 20 nm wide) with locally elevated concentrations of Ca2+ and Na+. The existence of such “functional” compartments with transient high ionic concentrations has been postulated for explaining the nigh efficiency of signal transmission during excitation-contraction coupling. We present results showing that electron probe microanalysis (EPMA) at the high spatial resolution of scanning transmission electron microscopy (STEM) can detect changes in sodium and calcium concentration of membrane-limited organelles and functional microdomains provided the cardiac myocytes are rapidly frozen at defined times during activation of contraction.

Hypoxia induced by Na 2S 2O 4 increases [Na +] i in mouse glomus cells, an effect depressed by cobalt. Experiments with Na +-selective microelectrodes and voltage-clamping

The intracellular sodium concentration ([Na +] i) and resting potential ( E m) of cultured mouse glomus cells (clustered and isolated) were simultaneously measured with intracellular Na +-sensitive and conventional, KCl-filled, microelectrodes. Results obtained in clustered and isolated cells were similar. During normoxia (PO 2 122 Torr), [Na +] i was 12–13 mM corresponding to a Na + equilibrium potential ( E Na) of about 58 mV. Em was about −42 mV. Hypoxia, induced by Na 2S 2O 4 1 mM (PO 2 10 Torr), depolarized the cells by about 20 mV, [Na +] i increased by 21 mM and E Na dropped to about 35 mV. One millimolar of CoCl 2 depressed, or blocked, the effects of Na 2S 2O 4 on [Na +] i but did not affect hypoxic depolarization. Voltage-clamping at −70 mV, while delivering pulses of different amplitudes, produced only small (about 10 pA) and slow TTX-insensitive inward currents. Fast and large (TTX-sensitive) inward currents were not detected. The cell conductance (measured with voltage ramps) was less than 1 nS. It was not affected by hypoxia but was depressed by cobalt. Voltage ramps elicited small inward currents in control and hypoxic solutions that were much smaller than those induced by barium (presumably enhancing calcium currents). Also, normoxic and hypoxic currents had lower thresholds and their troughs were at more negative voltages than in the presence of Ba 2+. All currents were blocked by 1 mM CoCl 2 suggesting that, at this concentration, cobalt exerted a nonspecific effect on glomus membrane channels. Hypoxia induced a large [Na +] i increase (presumably through inflow), but very small voltage-gated inward currents. Thus, Na + increases (inflow) probably occurred by disturbing a Na +/K + exchange mechanism and not by activation of voltage-gated channels.

The receptor subtype mediating the action of angiotensin II on intracellular sodium in rat proximal tubules.

1. An investigation was undertaken to explore the subtype of receptor involved in mediating the actions of angiotensin II on intracellular sodium content in suspensions of isolated proximal tubules of the rat. 2. Intracellular sodium content of the proximal tubules was measured with 23Na n.m.r. spectroscopy and under these conditions basal sodium content of the tubular cells was 69.04+/-1.73 nmol mg(-1) dry weight and the ATP levels, at 8.3+/-0.9 nmol ATP mg(-1) protein, were consistent with active respiration by the tissue. 3. In the presence of 10(-4) M PD123319, a selective non-peptide AT2 receptor antagonist, intracellular sodium levels rose from steady state by 30% (P < 0.01; n = 7) within 10 min of exposure to angiotensin II 10(-11) M. Over the subsequent 30 min steady state levels were re-established. Administration of angiotensin II 10(-11) M, in the presence of the selective AT1 receptor antagonist, losartan at either 10(-6) M (n = 5) or 10(-4) M (n = 6), was without effect on intracellular sodium levels, which were significantly different (P < 0.001) from those observed when PD 123319 was present. 4. Angiotensin II 10(-5) M, administered to the tubular suspension in the presence of 10(-4) M PD123319, decreased (P < 0.01, n = 6) intracellular sodium content by 16% in the first 5 min, but in the following 25 min returned to steady state levels. However, in the presence of losartan 10(-4) M, angiotensin II 10(-5) M had no effect on intracellular sodium content which was markedly different (P < 0.001) from that obtained in the presence of PD123319. 5. These findings show that at both the high and low concentrations of angiotensin II, its modulation of intracellular sodium levels within the proximal tubule cells is mediated via the activation of AT1 receptors. The intracellular mechanism underlying this effect remain to be investigated.

Hyperpolarization induces a rise in intracellular sodium concentration in dopamine cells of the substantia nigra pars compacta.

We investigated the effect of changes in membrane-voltage on intracellular sodium concentration ([Na+]i) of dopamine-sensitive neurons of the substantia nigra pars compacta in a slice preparation of rat mesencephalon. Whole-cell patch-clamp techniques were combined with microfluorometric measurements of [Na+]i using the Na+-sensitive probe, sodium-binding benzofuran isophthalate (SBFI). Hyperpolarization of spontaneously active dopamine neurons (recorded in current-clamp mode) caused the cessation of action potential firing accompanied by an elevation in [Na+]i. In dopamine neurons voltage-clamped at a holding potential of -60 mV elevations of [Na+]i were induced by long-lasting (45-60 s) voltage jumps to more negative membrane potentials (-90 to -120 mV) but not by corresponding voltage jumps to -30 mV. These hyperpolarization-induced elevations of [Na+]i were depressed during inhibition of I(h), a hyperpolarization-activated inward current, by Cs+. Hyperpolarization-induced elevations in [Na+]i might occur also in other cell types which express a powerful I(h) and might signal lack of postsynaptic activity.

Alpha-Adrenergic effects on Na+-K+ pump current in guinea-pig ventricular myocytes.

1. The whole-cell patch clamp was employed to study Na+-K+ pump current (Ip) in acutely isolated myocytes. alpha-Adrenergic receptors were activated with noradrenaline (NA) after blocking beta-adrenergic receptors with propranolol. Ip was measured as the current blocked by strophanthidin (Str). 2. Activation of alpha-receptors by NA increased Ip in a concentration-dependent manner. The K0.5 depended on intracellular calcium ([Ca2+]i), however maximal stimulation did not. At 15 nM [Ca2+]i the K0.5 was 219 nM NA whereas at 1.4 microM [Ca2+]i it was 3 nM. 3. The voltage dependence of Ip was not shifted by NA at either high or low [Ca2+]i. At each voltage, maximal stimulation of Ip was 14-15 %. 4. Staurosporine (St), an inhibitor of protein kinase C (PKC), eliminated the alpha-receptor-mediated stimulation of Ip at either high or low[Ca2+]i. 5. The stimulation of Ip was independent of changes in intracellular sodium or external potassium concentrations, and did not reflect a change in affinity for Str. 6. Phenylephrine, methoxamine and metaraminol, three selective alpha1-adrenergic agonists, stimulate Ip in a similar manner to NA. Stimulation of Ip by NA was eliminated by prazosin, an alpha1-antagonist, but was unaffected by yohimbine, an alpha2-antagonist. 7. We conclude noradrenaline activates ventricular alpha1-receptors, which are specifically coupled via PKC to increase Na+-K+ pump current. The sensitivity of the coupling is [Ca2+]i dependent, however the maximal increase in pump current is [Ca2+]i and voltage independent.

Co(III)EDTA as extra-cellular marker in μPIXE-analysis of rat cardiomyocytes

In previous studies no clear difference was found between the intra- and extra-cellular compartment in nuclear microprobe elemental distribution maps of freeze-dried cryo sections of heart tissue. Probably due to artefacts during the preparation of these samples, the intra-cellular and the extra-cellular content of elements are mixed up. In this article a method, using NaCo(III)EDTA as an extra-cellular marker, was applied to deconvolute the total ion content in an extra- and intra-cellular contribution. This method was both applied to normoxic heart tissue and low-flow ischemic heart tissue. Intra-cellular ion concentrations calculated from the corrected ion contents of the normoxic tissue agrees well with literature values. Moreover a clear elevation of the intra-cellular sodium and chlorine concentration was found in low-flow ischemic tissue.

Effects of external saccharides on cell wall properties and ion transport inHydrodictyon reticulatum

Various saccharides, when present at osmotically insignificant concentrations in growth media, were tested as to their effects on the cell walls of the green algaHydrodictyon reticulatum, manifesting themselves in differences in cell water and ion contents. BothD: -xylose andD: -mannose reduce the cell water content andD: -galactose does occasionally the same but onlyD: -xylose reduces significantly the intracellular sodium concentration, presumably by forming steric hindrances at the outlets of the sodium pumps at the outer surface of the cell membrane. No significant effects of eitherL: -arabinose orD: -arabinose on the cell water and ion contents were found.

Relationship Between Salt And Blood Pressure In Hypertensive Patients On Chronic ACE-Inhibition

We investigated the effect of a 4-day oral salt load (150 mmol NaCl extra per day) on blood pressure, erythrocyte sodium transport and the activity in the renin-angiotensin system in six males with primary hypertension, who had attained normotension on chronic enalapril treatment for 4 years. The design was a placebo-controlled, randomized, two-way cross over, double-blind study, i.e. each patient served as his own control. Intracellular erythrocyte sodium and potassium content were measured by flame photomometry. The increase in the intracellular sodium concentration during 1 h in 37 degrees C incubation of whole-blood with ouabain (compared with no-ouabain) was measured to determine the rate of active sodium efflux. 24-h blood pressure registration was performed with Space-lab equipment (SL 90202) before and at the end of the salt load. Left ventricular morphology was evaluated with echocardiography and the minimal vascular resistance of the hand vascular bed with water plethysmography at baseline and after 4 years on enalapril. Four years' enalapril treatment caused a significant decrease in blood pressure, left ventricular mass and minimal vascular resistance. During the 4-day salt load average 24-h blood pressure was significantly elevated, 129+/-3/85+/-2 mmHg as compared to 124+/-2/82+/-2 mmHg during placebo treatment (p=0.025). The change (delta) in MAP during high salt intake showed a negative relationship to delta-sodium efflux rate constant (r=-0.65, p=0.047). No significant relationship was found between the blood pressure response to the salt load and structural cardiovascular changes. In conclusion, a short-term oral salt load in hypertensive patients on chronic enalapril treatment caused a blood pressure rise, which was related to cellular sodium transport but not to structural cardiovascular changes.

Efflux of gamma-aminobutyric acid caused by changes in ion concentrations and cell swelling simulating the effect of cerebral ischaemia.

The relationships among ischaemic GABA efflux from brain tissue and extracellular and intracellular concentrations of sodium, chloride and potassium ions were investigated by means of 1) transverse hippocampal slices from rat and 2) functional expression of a high affinity GABA transporter in Xenopus oocytes. Brain slices were incubated for 20 min in medium where extracellular sodium and chloride were substituted with impermeant ions. Isethionate (Iseth) substitution for chloride generated a 7-fold increase in GABA efflux. Choline (Chol) but not N-methyl-D-glucamine (NMDG) substitution for sodium likewise increased GABA efflux. Reducing the osmolarity of the medium by decreasing both sodium and chloride concentrations (Hyp) increased GABA efflux 3-fold. This release was blocked by mannitol (Man). Blocking sodium channels with 1 microM of tetrodotoxin (TTX) also increased the release 3-fold. Energy deprivation (ED) increased the GABA release 50-fold. ED/Iseth left the release unchanged, ED/Chol increased the GABA efflux by 23%, whereas ED/NMDG reduced the release by 41%. Adding mannitol did not block the ED-evoked release, whereas TTX reduced it by 52%. Release of preloaded [3H]-GABA from oocytes expressing the GAT-1 GABA transporter was then examined. Depolarisation by current injection or 100 mM extracellular K+ did not increase GABA release. Sodium chloride injection, however, caused membrane depolarisation and a 100-fold increased GABA efflux from the oocytes. This release was blocked when the osmolarity was increased extracellularly by adding mannitol. These results show that 1) TTX releases GABA from brain tissue but blocks release during ED, 2) the high affinity GABA carrier must be altered in order to reverse, 3) ischaemic GABA release is sodium independent, and is modulated by large cations, 4) mannitol blocks the reversal of high affinity carriers in oocytes, but the release from brain slices during ED is unaffected. Taken together, the results suggest that ischaemic release of GABA from brain tissue does not occur by means of reversed high affinity carriers alone, but rather that it is controlled by more complex mechanisms.

Response of myocardial cellular energy metabolism to variation of buffer composition during open-chest experimental cardiopulmonary resuscitation in the pig.

The aim of the present study was to investigate possible relationships in piglets between myocardial energy-related metabolites and intracellular electrolytes during open-chest cardiopulmonary resuscitation (OCCPR) supplemented by the administration of alkaline buffers with varying sodium content. Our hypothesis was that an increasing myocardial intracellular sodium content would decrease the intracellular energy stores. In addition to haemodynamics, acid-base and blood gas variables were analysed, and myocardial biopsies were collected before and during OCCPR as well as after the return of spontaneous circulation. After a period of 4 min of untreated ventricular fibrillation (VF). 25 piglets were randomly allocated to one of four groups: OCCPR with normal saline (n = 5); OCCPR with sodium bicarbonate (SB) (n = 7); OCCPR with Tris buffer mixture (TBM) (n = 7); and a totally untreated control group (n = 6). The results showed that 4 min of untreated VF almost eradicated creatine phosphate (CrP) and that the ATP/ADP ratio decreased to 1.5-2.0. During OCCPR with normal saline, the myocardial content of CrP increased, whereas lactate, ATP and ADP levelled off and AMP decreased, causing an increased ATP/ADP ratio. The adenosine and inosine contents increased, whereas inosine monophosphate was unchanged at a low level, the adenosine and inosine contents being inversely correlated with the total content of adenine nucleotides. In both buffered groups, the increase in most energy-related metabolites (CrP, ATP, ADP, AMP and the ATP/ADP quotient) was less and in lactate more pronounced than in the group not being buffered, with no difference between the groups receiving SB or TBM. Although the intracellular potassium content was unaltered, the sodium, chloride and calcium concentration increased, more so in the group receiving SB. The intracellular content of sodium was correlated with that of calcium. Thus, buffering increased the myocardial AMP degradation during OCCPR by increasing the flux via the 5'-nucleotidase reaction, and SB increased the intracellular contents of sodium and calcium to a greater extent than did TBM.

NMR relaxation studies on hepatic intracellular and extracellular sodium in rats with burn injury.

In vivo longitudinal (T1) and transverse (T2) relaxation times of hepatic intracellular and extracellular sodium were studied in rats with sham burn or burn injury with 23Na NMR spectroscopy and shift reagent. Burn injury decreases hepatic extracellular sodium content by 17% compared with sham burn, whereas it increases the percent of the fast T2 component of extracellular sodium, suggesting an increase in the fraction of bound Na+/- sites in the extracellular space. It is interesting that the relaxation characteristics of intracellular sodium remained unchanged despite a 57% increase in intracellular sodium content, suggesting the increase in intracellular free sodium is matched by either a proportional increase in intracellular bound sodium or an uncovering of ordered domain sites that can preferentially orient rapidly exchanging sodium ions. This study also demonstrated that spin lattice (T1) relaxation rates or the percent contribution of the fast/slow T2 components of the combined intracellular/extracellular 23Na signal (before the infusion of shift reagent) may also be sensitive to changes in intracellular sodium levels during pathologic changes.

Ion homeostasis in brain cells: differences in intracellular ion responses to energy limitation between cultured neurons and glial cells

Intracellular concentrations of sodium, potassium and calcium together with membrane potentials were measured in cultured murine cortical neurons and glial cells under conditions which mimicked in vivo hypoxia, ischemia and hypoglycemia. These included; glucose omission with and without added pyruvate, addition of rotenone in the presence and absence of glucose and substitution of 2-deoxyglucose for glucose with and without rotenone. Cellular energy levels ([ATP], [ADP], [phosphocreatine], [creatine]) were measured in suspensions of C6 cells incubated in parallel under identical conditions. [Na +] i and [Ca 2+] i rose while [K +] i fell and plasma membrane depolarized when energy production was limited. Intracellular acidification was observed when glycolysis was the sole source for ATP synthesis. There was a positive correlation between the extent of energy depletion in glial cells and the magnitude and velocity of alterations in ion levels. Neither glycolysis alone nor oxidative phosphorylation alone were able to ensure unaltered ion gradients. Since oxidative phosphorylation is much more efficient in generating ATP than glycolysis, this finding suggests a specific requirement of the Na pump for ATP generated by glycolysis. Changes in [Na +] i and [K +] i observed during energy depletion were gradual and progressive whereas those in [Ca 2+] i were initially slow and moderate with large elevations occurring only as a late event. Increases in [Na +] i were usually smaller than reductions in [K +] i, particularly in the glia, suggestive of cellular swelling. Glia were less sensitive to identical insults than were neurons under all conditions. Results presented in this study lead to the conclusion that the response to energy deprivation of the two main types of brain cells, neurons and astrocytes, is a complex function of their capacity to produce ATP and the activities of various pathways which are involved in ion homeostasis.

Effects of sodium and potassium concentrations and pH on equine erythrocyte volume and deformability

During strenuous exercise, equine erythrocyte deformability is transiently decreased. Decreased deformability is associated with increased cell volume, decreased cell density and increased intracellular sodium, potassium and chloride concentrations. To better understand these changes, we attempted to reproduce exercise-associated changes in erythrocytes in vitro by adjusting plasma sodium, potassium and pH to levels which occur during racing activity. Increasing plasma sodium to 145 meq/1 and plasma potassium to 8 meq/1 resulted in decreased erythrocyte filterability, increased cell volume, and increased intracellular sodium, potassium and chloride. Incubation of erythrocytes with frusemide and bumetanide, but not ouabain or [(dihydroindenyl)oxy]alkanoic acid (DIOA), attenuated these changes. Decreasing plasma pH to 7.0 also decreased erythrocyte filterability, increased cell volume, and increased intracellular concentrations of sodium, potassium and chloride. Ouabain, but not frusemide or bumetanide, prevented the decrease infilterability and attenuated the increase in intracellular sodium. Addition of DIOA exacerbated the effect of pH on erythrocyte filterability. Therefore, exercise-associated changes in erythrocyte deformability, size and electrolyte concentration can be reproduced in vitro by increasing plasma sodium and potassium concentrations and by decreasing pH.

Phorbol 12-myristate 13-acetate down-regulates Na,K-ATPase independent of its protein kinase C site: decrease in basolateral cell surface area.

The effect of protein kinase C (PKC) stimulation on the pump current (Ip) generated by the Na,K-ATPase was measured in A6 epithelia apically permeabilized with amphotericin B. Phorbol 12-myristate 13-acetate (PMA) produced a decrease in Ip carried by sodium pumps containing the endogenous Xenopus laevis or transfected Bufo marinus alpha 1 subunits (approximately 30% reduction within 25 min, maximum after 40 min) independent of the PKC phosphorylation site (T15A/S16A). In addition to this major effect of PMA, which was independent of the intracellular sodium concentration and was prevented by the PKC inhibitor bisindolylmaleimide GF 109203X (BIM), another BIM-resistant, PKC site-independent decrease was observed when the Ip was measured at low sodium concentrations (total reduction approximately 50% at 5 mM sodium). Using ouabain binding and cell surface biotinylation, stimulation of PKC was shown to reduce surface Na,K-ATPase by 14 to 20% within 25 min. The same treatment stimulated fluid phase endocytosis sevenfold and decreased by 16.5% the basolateral cell surface area measured by transepithelial capacitance measurements. In conclusion, PKC stimulation produces a decrease in sodium pump function which can be attributed, to a large extent, to a withdrawal of sodium pumps from the basolateral cell surface independent of their PKC site. This reduction of the number of sodium pumps is parallel to a decrease in basolateral membrane area.