L-Fucose Is a Potent Inhibitor of myo-Inositol Transport and Metabolism in Cultured Neuroblastoma Cells

It has been proposed that abnormal myo-inositol metabolism may be a factor in the development of diabetic complications. Studies with animal models of diabetes and cultured cells have suggested that hyperglycemia by an unknown mechanism may alter myo-inositol metabolism and content. Recently, we have shown that L-fucose, a 6-deoxy sugar whose content has been reported to be increased in diabetes, is a potent inhibitor of myo-inositol transport. To examine the effect of L-fucose on myo-inositol metabolism, neuroblastoma cells were cultured in medium supplemented with L-fucose. L-Fucose is a competitive inhibitor of Na(+)-dependent, high-affinity myo-inositol transport. The Ki for inhibition of myo-inositol transport by L-fucose is about 3 mM. L-Fucose is taken up and accumulates in neuroblastoma cells. The uptake of L-fucose is inhibited by Na+ depletion, D-glucose, glucose analogues, phloridzin, and cytochalasin B. In contrast, neither myo-inositol nor L-glucose inhibits L-fucose uptake. Chronic exposure of neuroblastoma cells to 1-30 mM L-fucose causes a decrease in myo-inositol accumulation and incorporation into inositol phospholipids, intracellular free myo-inositol content, and phosphatidylinositol levels. Na+,K(+)-ATPase transport activity is decreased by about 15% by acute or chronic exposure of neuroblastoma cells to L-fucose. Similar defects occur when neuroblastoma cells are exposed chronically to 30 mM glucose. Cell myo-inositol metabolism and Na+/K(+)-pump activity are maintained when 250 microM myo-inositol is added to the L-fucose-supplemented medium. Unlike the effect of chronic exposure of neuroblastoma cells to medium containing 30 mM glucose, the resting membrane potential of neuroblastoma cells is not altered by chronic exposure of the cells to 30 mM L-fucose. The effect of L-fucose on cultured neuroblastoma cell properties occurs at concentrations of L-fucose which may exist in the diabetic milieu. These data suggest that increased concentrations of L-fucose may have a role in myo-inositol-related defects in mammalian cells.     

Effect of Sorbinil on myo-Inositol Metabolism in Cultured Neuroblastoma Cells Exposed to Increased Glucose Levels

Neuroblastoma cells were used to determine the effect of sorbinil on myo-inositol metabolism in cells exposed to elevated levels of glucose in culture. Exposing cells to elevated levels of glucose led to an increase in levels of intracellular sorbitol. The increase in sorbitol levels was dependent on the extracellular glucose concentration. In contrast, the myo-inositol content of cells was decreased in the presence of increasing concentrations of extracellular glucose. Increasing the concentration of glucose in the culture medium caused a decrease in myo-inositol uptake and in the incorporation of extracellular myo-inositol into phospholipid. The effect of elevated glucose levels on myo-inositol metabolism and sorbitol accumulation was blocked by addition of 0.4 mM sorbinil. The ability of sorbinil to block the decrease in myo-inositol metabolism and sorbitol accumulation caused by 30 mM extracellular glucose was dependent on its concentration. Maximal effects were obtained with 0.4 mM sorbinil. However, there was some variation in the degree of effectiveness among batches of sorbinil. These results at the cellular level suggest that the intracellular accumulation of sorbitol is responsible for the alteration of myo-inositol metabolism observed in neuroblastoma cells exposed to elevated glucose concentrations.     

Effect of Fructose Supplementation on Sorbitol Accumulation and myolnositol Metabolism in Cultured Neuroblastoma Cells Exposed to Increased Glucose Concentrations

Aldose reductase activity is increased in neuroblastoma cells grown in media containing 30 mM fructose and/or 30 mM glucose. Neuroblastoma cells cultured in media supplemented with increased concentrations of glucose and fructose amass greater amounts of sorbitol than do cells exposed to media containing only high glucose concentrations. The increase in sorbitol content is dependent on the fructose and glucose concentration in the media. The increase in sorbitol content caused by exposing neuroblastoma cells to media containing 30 mM glucose/30 mM fructose is due to a protein synthesis sensitive mechanism and not to an alteration in the redox state. The addition of sorbinil to media containing 30 mM glucose blocks the increase in sorbitol content. In contrast, sorbinil treatment of media containing 30 mM glucose/30 mM fructose does not totally block the increase in sorbitol levels. myo-Inositol accumulation and incorporation into inositol phospholipids and intracellular myo-inositol content are decreased in cells chronically exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose compared to cells cultured in unsupplemented media or media containing 30 mM fructose. However, maximal depletion of myo-inositol accumulation and intracellular content occurs earlier in cells exposed to media containing 30 mM glucose/30 mM fructose than in cells exposed to media supplemented with 30 mM glucose. Sorbinil treatment of media containing 30 mM glucose/30 mM fructose maintains cellular myo-inositol accumulation and incorporation into phospholipids at near normal levels. myo-Inositol content in neuroblastoma cells chronically exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose recovers within 72 h when the cells are transferred to unsupplemented media or media containing 30 mM fructose. In contrast, the sorbitol content of cells previously exposed to media containing 30 mM glucose or 30 mM glucose/30 mM fructose then transferred into media containing 30 mM fructose remains elevated compared to the sorbitol content of cells transferred into unsupplemented media. These data suggest that fructose may be activating or increasing sorbinil-resistant aldose reductase activity as well as partially blocking sorbitol dehydrogenase activity. The presence of increased concentrations of fructose in combination with increased glucose levels may enhance alterations in cell metabolism and properties due to increased sorbitol levels.     

The hormone-sensitive hepatic Na+-pump. Evidence for regulation by diacylglycerol and tumor promoters

Ouabain-sensitive 86Rb+ uptake by isolated rat hepatocytes was studied to elucidate how Ca2+-mobilizing hormones stimulate the Na+-pump. Stimulation of this uptake was observed with concentrations of vasopressin ([8-arginine]vasopressin, AVP), angiotensin II, and norepinephrine which elicited Ca2+ mobilization and phosphorylase activation. These results suggested that changes in cytosolic Ca2+, mediated by inositol trisphosphate, might trigger sodium pump stimulation by AVP. However, in hepatocytes incubated in Ca2+-free Krebs-Henseleit buffer, Na+-pump activity was not altered over 15 min by either 1.5 mM EGTA or 1.5 mM Ca2+. Furthermore, incubation of cells in 5 mM EGTA for 15-30 min drastically impaired the ability of AVP to increase cytosolic Ca2+, but only modestly attenuated AVP-stimulated Na+-pump activity. Two tumor promoters, phorbol myristate acetate (PMA) and mezerein, stimulated Na+/K+-ATPase-mediated transport activity. Similarly, addition of synthetic diacylglycerols or of exogenous phospholipase C from Clostridium perfringens to increase endogenous diacylglycerol levels also resulted in a stimulation of the Na+-pump in the absence of changes in cytosolic or total cellular Ca2+ levels. Stimulation of the Na+-pump by the combination of maximal concentrations of PMA and AVP did not produce an additive response, and both agents displayed a transient time course, suggesting that the two agents share a common mechanism. Stimulation of the Na+-pump by AVP and PMA was not blocked by amiloride analogs which inhibit Na+/H+ exchange, but these compounds blocked the action of insulin. These data suggest that the elevated Na+/K+-ATPase-mediated transport activity observed in hepatocytes following exposure to Ca2+-mobilizing hormones is a consequence of stimulated diacylglycerol formation and may involve protein kinase C.     

Retinoic-acid-induced differentiation of HL-60 cells is associated with biphasic activation of the Na+− K+ pump

The human promyelocytic leukemia cell line, HL-60, can be induced to differentiate into granulocyte-like cells when cultured in the presence of 10(-6) M retinoic acid (RA) for several days. Following the addition of RA two kinds of changes occur. First, there are early changes that comprise an increase in the intracellular concentration of sodium ions [Na]i, which reaches its maximum after 6 h, and an increase in the activity of the Na+-pump, which is reflected by an ouabain-sensitive K+ influx that peaks at 8 h (170% of the control value) and that occurs without any change in the number of pump molecules, as measured by the binding of 3H-ouabain. Second, beginning after 12 h of culture with RA, a decrease in the number of ouabain-binding sites occurs, this being accompanied by an increase in the number of K+ ions actively transported by each site. The effect of modulation of Na+-pump activity on the RA-induced differentiation of HL-60 cells was studied using low, noncytotoxic concentrations of ouabain which, although alone having no differentiating effect, accelerated and potentiated the effect of RA on differentiation. When added in combination, these drugs induced rapid stimulation of the Na+-pump, which reached its peak after 2 h. These results indicate that a concomitant increase in the level of [Na+]i and in the activity of the Na+-pump constitute primary events in the interaction between RA and HL-60 cells, and that cation fluxes may play a role in the initiation of the process of differentiation.     

Decreased intracellular Na+ concentration is an early event in murine erythroleukemic cell differentiation

A decrease in Na+/K+-pump activity is an early event of Friend murine erythroleukemic (MEL) cell differentiation along the erythroid pathway. This decreased Na+/K+-pump activity has been proposed to be an essential step in differentiation which would cause a rise in intracellular Na+ concentration and then, by means of Na+/Ca2+ exchange, an increase in intracellular Ca2+. An increase in intracellular Ca2+ has been proposed to be essential for induction of differentiation. A critical prediction of this Na+-Ca2+ hypothesis is the rise in intracellular Na+. To test this prediction we have measured intracellular Na+ using a novel triple isotope method involving 3H2O, [14C]sucrose, and 22Na to measure total water, extracellular fluid, and Na+, respectively. 22Na equilibration occurred in less than 10 min. In uninduced cells, intracellular Na+ was 15.2 +/- 2.2 mM (S.D., n = 22); after induction for 14-16 h with dimethyl sulfoxide, intracellular Na+ decreased significantly (p less than 0.0001) to 8.4 +/- 1.4 mM (n = 21). The time course of the decline in intracellular Na+ paralleled that of the decrease in the Na+/K+-pump activity. These results are in direct contradiction to the Na+-Ca2+ hypothesis and suggest that observed changes in Na+/K+-pump activity can be explained solely on the basis of changes in intracellular Na+. The drop in intracellular Na+ is due to a decrease in Na+ influx. We suggest, however, that the decrease in the Na+ influx is not itself an essential event of differentiation, but may be induced by a change in the flux of another ion coupled to Na+.     

myo-Inositol Metabolism in 41 A3 Neuroblastoma Cells: Effects of High Glucose and Sorbitol Levels

Neuroblastoma cells were used to determine the effect of high carbohydrate and polyol levels on myo-inositol metabolism. The presence of elevated concentrations of glucose or sorbitol caused a significant decrease in both inositol accumulation and incorporation into phospholipid. These conditions, however, did not alter the accumulation of the other phospholipid head groups or the growth rate and water content of the cells. Two weeks of growth in either of the modified conditions was necessary to obtain a maximal effect on inositol incorporation. In contrast, growth in elevated concentrations of fructose, mannitol, or dulcitol had no effect on inositol metabolism. The reduced inositol accumulation and incorporation into lipids seen with glucose or sorbitol supplementation resulted in a decrease in the total phosphatidylinositol content of the cell without changing the levels of the other phospholipids. Kinetic analysis of cells grown in the presence of elevated glucose indicated that V'max for inositol uptake was significantly decreased with little change in the K'm. These data suggest that glucose decreases myo-inositol uptake in this system by noncompetitive inhibition. Cells grown in the presence of increased glucose also had elevated levels of intracellular sorbitol and decreased levels of myo-inositol. These results suggest that the high levels of glucose and sorbitol which exist in poorly regulated diabetes may be at least partially responsible for diabetic neuropathy via a reduction in the cellular content of myo-inositol and phosphatidylinositol. This system may be a useful model to determine the effect of reduced inositol phospholipid levels on neural cell function.     

Nerve Growth Factor Induces Na+, K+-ATPase in a Nerve Cell Line

The effects of nerve growth factor (NGF) on induction of Na+,K+-ATPase were examined in a rat pheochromocytoma cell line, PC12h. Na+,K+-ATPase activity in a crude particulate fraction from the cells increased from 0.37 +/- 0.02 (n = 19) to 0.55 +/- 0.02 (n = 20) (means +/- SEM, mumol Pi/min/mg of protein) when cultured with NGF for 5-11 days. The increase caused by NGF was prevented by addition of specific anti-NGF antibodies. Epidermal growth factor and insulin had only a small effect on induction of Na+,K+-ATPase. A concentration of basic fibroblast growth factor three times higher than that of NGF showed a similar potency to NGF. The molecular form of the enzyme was judged as only the alpha form in both the untreated and the NGF-treated cells by a simple pattern of low-affinity interaction with cardiotonic steroids: inhibition of enzyme activity by strophanthidin (Ki approximately 1 mM) and inhibition of Rb+ uptake by ouabain (Ki approximately 100 microM). As a consequence, during differentiation of PC12h cells to neuron-like cells, NGF increases the alpha form of Na+,K+-ATPase, but does not induce the alpha(+) form of the enzyme, which has a high sensitivity for cardiotonic steroid and is a characteristic form in neurons.     

Effect of Monovalent Cations on Na+/Ca2+ Exchange and ATP-Dependent Ca2+ Transport in Synaptic Plasma Membranes

Two Ca2+ transport systems were investigated in plasma membrane vesicles isolated from sheep brain cortex synaptosomes by hypotonic lysis and partial purification. Synaptic plasma membrane vesicles loaded with Na+ (Na+i) accumulate Ca2+ in exchange for Na+, provided that a Na+ gradient (in leads to out) is present. Agents that dissipate the Na+ gradient (monensin) prevent the Na+/Ca2+ exchange completely. Ca2+ accumulated by Na+/Ca2+ exchange can be released by A 23187, indicating that Ca2+ is accumulated intravesicularly. In the absence of any Na+ gradient (K+i-loaded vesicles), the membrane vesicles also accumulate Ca2+ owing to ATP hydrolysis. Monovalent cations stimulate Na+/Ca2+ exchange as well as the ATP-dependent Ca2+ uptake activity. Taking the value for Na+/Ca2+ exchange in the presence of choline chloride (external cation) as reference, other monovalent cations in the external media have the following effects: K+ or NH4+ stimulates Na+/Ca2+ exchange; Li+ or Cs+ inhibits Na+/Ca2+ exchange. The ATP-dependent Ca2+ transport system is stimulated by increasing K+ concentrations in the external medium (Km for K+ is 15 mM). Replacing K+ by Na+ in the external medium inhibits the ATP-dependent Ca2+ uptake, and this effect is due more to the reduction of K+ than to the elevation of Na+. The results suggest that synaptic membrane vesicles isolated from sheep brain cortex synaptosomes possess mechanisms for Na+/Ca2+ exchange and ATP-dependent Ca2+ uptake, whose activity may be regulated by monovalent cations, specifically K+, at physiological concentrations.     

Regulation of Na+,K+ pump activity by nerve growth factor in chick embryo dorsal root ganglion cells

Nerve growth factor (NGF) is required for the growth and development of sensory and sympathetic neurons. Incubation of chick dorsal root ganglionic cells without NGF resulted in a decrease of active (Na+,K+-pump-mediated) K+ influx over a period of several hours. Addition of NGF to NGF-deprived cells caused 1) a return of the active K+ influx to the values occurring in cells continuously exposed to NGF, preceded by 2) a very rapid, but transient overstimulation of the Na+,K+-pump-mediated K+ influx. Restoration of normal Na+,K+-pump activity occurred at NGF concentrations of 1 biological unit/ml or greater, whereas the NGF concentration in the 1-100 biological unit/ml range affected the rapidity with which the pump restoration took place. The transient pump behavior was only observed in NGF-deprived cells and could not be elicited in NGF-supported steady-state cells or in cells having already received delayed NGF once. This transient Na+,K+-pump behavior was exclusively displayed in conjunction with a high intracellular Na+ concentration. Decreasing the external Na+ concentration below 70 mM reduced the hyperstimulation response to NGF, until at 10 mM Na+ the delayed presentation of NGF caused no overshoot at all. The effect of NGF on the Na+,K+-pump was specific for the NGF molecule and could not be mimicked by other proteins.     

Ouabain Binding, ATP Hydrolysis, and Na+,K+-Pump Activity During Chemical Modification of Brain and Muscle Na+,K+-ATPase

The effects of 16 group-specific, amino acid-modifying agents were tested on ouabain binding, catalytical activity of membrane-bound (rat brain microsomal), sodium dodecyl sulfate-treated Na+,K(+)-ATPase, and Na+,K(+)-pump activity in intact muscle cells. With few exceptions, the potency of various tryptophan, tyrosine, histidine, amino, and carboxy group-oriented drugs to suppress ouabain binding and Na+,K(+)-ATPase activity correlated with inhibition of the Na+,K(+)-pump electrogenic effect. ATP hydrolysis was more sensitive to inhibition elicited by chemical modification than ouabain binding (membrane-bound or isolated enzyme) and than Na+,K(+)-pump activity. The efficiency of various drugs belonging to the same "specificity" group differed markedly. Tyrosine-oriented tetranitromethane was the only reagent that interfered directly with the cardiac receptor binding site as its inhibition of ouabain binding was completely protected by ouabagenin preincubation. The inhibition elicited by all other reagents was not, or only partially, protected by ouabagenin. It is surprising that agents like diethyl pyrocarbonate (histidine groups) or butanedione (arginine groups), whose action should be oriented to amino acids not involved in the putative ouabain binding site (represented by the -Glu-Tyr-Thr-Trp-Leu-Glu- sequence), are equally effective as agents acting on amino acids present directly in the ouabain binding site. These results support the proposal of long-distance regulation of Na+,K(+)-ATPase active sites.     

Effect of Fatty Acids on [3H]Ouabain Binding to Cerebromicrovascular (Na++ K+)-ATPase

The effects of short- and long-chain fatty acids on the cerebromicrovascular (Na+ + K+)-ATPase were investigated using specific [3H]ouabain binding to the enzyme. Specific binding increased linearly with total microvessel protein (37-110 micrograms) and was time-dependent with maximum binding obtained by 10 min. Arachidonic acid, but not palmitic acid, stimulated [3H]ouabain binding in a dose-dependent manner, with a 105% increase over basal levels at 100 microM arachidonic acid. Preincubation of the microvessels with arachidonic acid did not alter the stimulation observed. 4-Pentenoic acid stimulated [3H]ouabain binding only at high concentrations (10 mM). Scatchard analysis of [3H]ouabain binding to untreated microvessels yielded a single class of "high-affinity" binding sites with an apparent binding affinity (KD) of 64.7 +/- 2.0 nM and a binding capacity (Bmax) of 10.1 +/- 1.5 pmol/mg protein. In the presence of 100 microM arachidonic acid, a monophasic Scatchard plot also was obtained, but the KD significantly decreased to 51.9 +/- 2.7 nM (p less than 0.01), whereas the Bmax remained virtually unchanged (12.5 +/- 1.2 pmol/mg protein). The stimulation of [3H]ouabain binding in the presence of arachidonic acid was potentiated by 4-pentenoic acid, but not by indomethacin or eicosatetraynoic acid. These data suggest that long-chain polyunsaturated fatty acids may be involved in the regulation of blood-brain barrier (Na+ + K+)-ATPase and may play a role in the cerebral dysfunction associated with diseases in which plasma levels of nonesterified fatty acids are elevated.     

Elevated Levels of Glucose and L-Fucose Reduce 22Na+ Uptake and Whole Cell Na+ Current in Cultured Neuroblastoma Cells

Abstract: Na⁺ flux was studied in cultured neuroblastoma cells grown in medium containing increased glucose or L - fucose concentrations. Chronic exposure of neuroblastoma cells to 30 m M glucose or 30 m M L-fucose caused a decrease in ouabain-sensitive and veratridine-stimulated ²²Na⁺ uptake compared with cells cultured in unsupplemented medium. The Na⁺ current, determined by using whole-cell configuration of the patch clamp, was also decreased in these cells. Tetrodotoxin (3 μ M ), which blocked whole cell Na⁺ currents, also blocked veratridine-stimulated ²²Na⁺ accumulation. Culturing cells in medium containing 30 m M fructose as an osmotic control had no effect on Na⁺ flux. Specific [³H] saxitoxin binding was not affected by 30 m M glucose or 30 m M L-fucose compared with cells grown in unsupplemented medium, suggesting that the number of Na⁺ channels was not decreased. These studies suggest that exposing cultured neuronal cells to conditions that occur in the diabetic milieu alters Na⁺ transport and Na⁺-channel activity.     

The effect of divalent cations on Na+ tolerance in Charophytes. I: Char a buckellii

Abstract The comparative Na⁺ tolerance of Chora buckellii cultured in freshwater (FW) or artificial Waldsea water (AWW, which contains about 110 mol m^?3 each Na ⁺, Mg²⁺, Cl^? and SO^2-₄ was tested with respect to the external Na⁺ to Ca²⁺ ratio (Na: Ca). Fifty per cent of FW cells subjected to 70 mol m^?3 NaCl, which raised Na:Ca from 10: 1 to 700: 1 and the external osmotic pressure from 0.024 to 0.402 MPa, died within 6 d. Death was associated with the loss of Na/K selectivity, H⁺ -pump activity and turgor. Restoration of Na:Ca to 10:1 in high Na⁺ medium with CaCl₂ ensured 100% survival and maintained H⁺-pump activity and Na/K selectivity of FW cells. Turgor was regulated within 3 d with net uptake of Na ⁺, K⁺ and Cl^? in the vacuolc. Mg²⁺ was not as effective as Ca²⁺ in enhancing survival or maintaining H⁺ -pump activity and Na/K selectivity of FW cells in the presence of elevated Na⁺. However, turgor was regulated within 3 d by accumulation of Cl^? and an unknown cation in the vacuole. All AWW cells subjected to an increase of 70 mol m ^?3 NaCl, which raised Na: Ca from 16:1 to 25: 1 and the external osmotic pressure from 0.915 to 1.22 MPa, survived and maintained H ⁺ -pump activity. Turgor was regulated within 6d by accumulating Na ⁺, K⁺ and Cl^? in the vacuole. All AWW cells subjected to 70molm^?3 NaCl in a medium in which Na:Ca was equal to 700:1 survived and maintained H ⁺ -pump activity, but showed loss of Na/K selectivity. Turgor was regulated with an unknown osmoticum(a) within 6 d.     

Cilostazol, a cyclic AMP phosphodiesterase inhibitor, stimulates nitric oxide production and sodium potassium adenosine triphosphatase activity in SH-SY5Y human neuroblastoma cells.

Deficiencies in cellular cyclic AMP (cAMP) and nitric oxide (NO) production are thought to be involved in the pathogenesis of diabetic neuropathy. We used a human neuroblastoma cell line, SH-SY5Y, to investigate the effect of cilostazol, a specific cAMP phosphodiesterase inhibitor, on NO production and Na+, K+-ATPase activity. SH-SY5Y cells were cultured under 5 or 50 mM glucose for 5-6 days, the cells were then exposed to cilostazol or other chemicals and nitrite, cAMP and Na+, K+-ATPase activity were measured. In cells grown in 50 mM glucose, cilostazol was observed to increase significantly both NO production and cellular cAMP accumulation in a time- and dose-dependent manner. Cilostazol also significantly recovered reduced levels of protein kinase A activity (PKA) in 50 mM glucose. Furthermore, a PKA inhibitor, H-89 significantly suppressed the increase in NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production by activating PKA. Cilostazol did not affect either sorbitol or myo-inositol concentrations. Dexamethasone, which is known to induce inducible NO synthase, had no effect on NO production stimulated by cilostazol, suggesting that cilostazol stimulates NO production catalyzed by neuronal constitutive NO synthase (ncNOS) in SH-SY5Y cells. L-arginine, which is an NO agonist enhanced Na+, K+-ATPase activity in cells grown in 50 mM glucose, NG-nitro-L-arginine methyl ester (L-NAME), which is an NOS inhibitor inhibited basal Na+, K+-ATPase activity in 5 mM glucose and suppressed the increased enzyme activity induced by cilostazol in 50 mM glucose. The above results confirmed our previous observation that NO regulates Na+, K+-ATPase activity in SH-SY5Y cells and suggest that cilostazol increases Na+, K+-ATPase activity, at least in part, by stimulating NO production. The present results also suggest that cilostazol has a beneficial effect on diabetic neuropathy by improving Na+, K+-ATPase activity via directly increasing cAMP and NO production in nerves.     

Acute changes in myo-inositol uptake and 22Na+ flux in murine neuroblastoma cells (N1E-115) following insulin

myo-Inositol uptake was investigated in a murine neuroblastoma clone (N1E-115) to determine the effect of altered Na+,K+-ATPase activity. The Na+ ionophore monensin, and veratridine, an alkaloid affecting voltage-dependent Na+ entry, increased acute 22Na+ uptake and 22Na+ efflux from pre-loaded cells, concomitant with enhanced myo-inositol uptake. This effect was also seen following insulin. Insulin-stimulated myo-inositol uptake was inhibited by amiloride, ouabain and pyrithiamine. Amiloride inhibition suggests that activation of Na+/H+ exchange preceding Na+,K+-ATPase activation is involved in insulin stimulation of myo-inositol uptake. Pyrithiamine inhibition is an indication of prior activation of the Na+,K+-ATPase alpha + catalytic subunit by insulin. The results provide evidence that insulin contributes to the maintenance of Na+,K+-ATPase in neuronal tissue.     

Mechanisms and consequences of Na+,K+-pump regulation by insulin and leptin.

Hormonal control of the Na+,K+-pump modulates membrane potential in mammalian cells, which in turn drives ion coupled transport processes and maintains cell volume and osmotic balance. Na+,K+-pump regulation is particularly important in the musculoskeletal, cardiovascular and renal systems. Decreased Na+,K+-pump activity can result in a rise in intracellular Na+ concentrations which in turn increase Na+/Ca2+ exchange, thereby raising intracellular Ca2+ levels. In cardiac and skeletal muscle, this could interfere with normal contractile activity. Similarly, in vascular smooth muscle the result would be resistance to vasodilation. Inhibition of the Na+,K+-pump can also reduce the driving force for renal tubular Na+ reabsorption, elevating Na+ excretion. By virtue of decreasing the membrane potential, thus allowing more efficient depolarization of nerve endings and by increasing intracellular Ca2+, inhibition of the Na+,K+-pump can increase nervous tone. The ability of insulin to stimulate the Na+,K+-pump in various cells and tissues, and the physiological significance thereof, have been well documented. Much less is known about the effect of leptin on the Na+,K+-pump. We have shown that leptin inhibits Na+,K+-pump function in 3T3-L1 fibroblasts. Defects in insulin and leptin action are associated with diabetes and obesity, respectively, both of which are commonly associated with cardiovascular complications. In this review we discuss the mechanisms of Na+,K+-pump regulation by insulin and leptin and highlight how, when they fail, they may contribute to the pathophysiology of hypertension associated with diabetes and obesity.     

Effects of Diltiazem on Bioenergetics, K+ Gradients, and Free Cytosolic Ca2+ Levels in Rat Brain Synaptosomes Submitted to Energy Metabolism Inhibition and Depolarization

Diltiazem was able to decrease the oxygen consumption rate and lactate production in synaptosomes isolated from rat forebrains, both under control and depolarized (40 microM veratridine) conditions, starting from a concentration of 250 microM. This effect was particularly evident when synaptosomes were depolarized by veratridine. This depolarization-counteracting action was evident also when transplasma membrane K+ diffusion potentials were measured after depolarization induced by veratridine and by rotenone with a glucose shortage. The concentrations of ATP, phosphocreatine, and creatine were less sensitive to diltiazem action. The concentration/response relationships were the same as those found for the oxygen consumption were the same as those found for the oxygen consumption rate, lactate production, and K+ diffusion potentials. The effects of 0.5 mM diltiazem in counteracting inhibition of energy metabolism induced by rotenone without glucose were no longer detectable when either Ca2+ or Na+ was absent from the incubation medium of synaptosomes. Diltiazem at the same concentrations (starting from 250 microM) was able to inhibit both the veratridine-induced and the rotenone-without-glucose-induced increase in intrasynaptosomal free Ca2+ levels evaluated with the fluorescent probe quin2. The results are discussed in view of a possible effect of diltiazem on voltage-dependent Na+ channels and the possibility of utilizing this approach for counteracting neuronal failure due to derangement of energy metabolism or hyperexcitation.     

Effects of Systemic Kainic Acid Administration on Regional Na+, K+-ATPase Activity in Rat Brain

Changes in the activity of Na+,K+-ATPase and in the water, Na+, and K+ levels in the parietal cortex, hippocampus, and thalamus were investigated in rats 1, 3, 6, and 24 h following systemic kainic acid injection. An increase in Na+,K+-ATPase activity was observed in all three regions 3 h after the treatment, with a subsequent decrease in enzyme activity. The elevation in Na+,K+-ATPase activity was accompanied by an increase in the Na+ content and a decrease in the K+ content. These changes are presumed to occur because of repeated discharges and excessive prolonged depolarization in response to kainic acid. The decreases in Na+,K+-ATPase activity 6 and 24 h following kainic acid treatment coincide with neuropathological damage and edema formation, mainly in the hippocampus and thalamus.     

A Na+- and Cl- -activated K+ channel in the thick ascending limb of mouse kidney

This study investigates the presence and properties of Na+-activated K+ (K(Na)) channels in epithelial renal cells. Using real-time PCR on mouse microdissected nephron segments, we show that Slo2.2 mRNA, which encodes for the K(Na) channels of excitable cells, is expressed in the medullary and cortical thick ascending limbs of Henle's loop, but not in the other parts of the nephron. Patch-clamp analysis revealed the presence of a high conductance K+ channel in the basolateral membrane of both the medullary and cortical thick ascending limbs. This channel was highly K+ selective (P(K)/P(Na) approximately 20), its conductance ranged from 140 to 180 pS with subconductance levels, and its current/voltage relationship displayed intermediate, Na+-dependent, inward rectification. Internal Na+ and Cl- activated the channel with 50% effective concentrations (EC50) and Hill coefficients (nH) of 30 +/- 1 mM and 3.9 +/- 0.5 for internal Na+, and 35 +/- 10 mM and 1.3 +/- 0.25 for internal Cl-. Channel activity was unaltered by internal ATP (2 mM) and by internal pH, but clearly decreased when internal free Ca2+ concentration increased. This is the first demonstration of the presence in the epithelial cell membrane of a functional, Na+-activated, large-conductance K+ channel that closely resembles native K(Na) channels of excitable cells. This Slo2.2 type, Na+- and Cl--activated K+ channel is primarily located in the thick ascending limb, a major renal site of transcellular NaCl reabsorption.