Effects of sodium fluxes and ouabain on brain-slice tryptophan transport

Efflux of preloaded [3H]tryptophan from rat cerebral cortex slices has been monitored into superfusion media that were altered in their sodium content. Total replacement of sodium with choline greatly increased the release of tryptophan. This release could be cancelled by re-introducing sodium into the slices. A brief exposure to ouabain, an efficient inhibitor of Na+, K+-ATPase activity, only slightly increased tryptophan efflux at the concentration of 0.1 mM, whereas at 1.0 mM it produced a similar effect as the sodium-free medium. Accordingly, when the slices were superfused in the presence of ouabain and sodium, the change of medium to sodium-free caused much greater relative enhancement of tryptophan efflux with 0.1 than 1.0 mM ouabain. Tryptophan efflux was modified by changes in sodium fluxes also in slices initially depleted of sodium ions and treated with ouabain. The results suggest that the sodium-free medium and ouabain have a similar mechanism of action in modifying the tryptophan transport, and that the cation gradients across the cell membranes are more crucial for normal amino acid transport than the functional Na+, K+-ATPase.     

Na+-K+-ATPase inhibition stimulates PGE release in guinea pig taenia coli.

Inhibition of the plasma membrane enzyme Na+-K+-ATPase by ouabain zero extracellular K+, or low extracellular Na+, markedly augmented prostaglandin E release from the guinea pig taenia coli. Data suggest this phenomenon may be linked directly to Na+-K+-ATPase or Na+ pump activities, or changes in intracellular K+ concentration. The augmented prostaglandin E release was not due to changes in intracellular Na+, Ca2+, pH, or membrane potential, resulting from Na+ pump inhibition. The characteristics of the plasma membrane may exert a control on prostaglandin E release in this smooth muscle.     

Renal Na+-K+-ATPase in renin release

The effects of ouabain and furosemide on renin secretion, renal function, and renal Na+-K+-ATPase were investigated in anesthetized dogs. Furosemide (2 mg/kg) induced significant diuresis, natriuresis, an increase in renal blood flow (RBF), and a fivefold increase in renin secretory rate (RSR), but no changes in glomerular filtration rate (GFR). Infusion of ouabain (1 microgram . kg-1 . min-1) into one renal artery during furosemide diuresis increased fractional sodium excretion from 22 +/- 2 to 30 +/- 3% from the ipsilateral kidney but did not change urine flow, RBF, or GFR, whereas RSR fell to control values (698 +/- 203 to 137 +/- 43). When ouabain preceded furosemide, the rise in RBF and RSR induced by furosemide was abolished but sodium excretion increased. Ouabain infused in vivo inhibited Na+-K+-ATPase in microsomal fractions from cortex (34%) and medulla (27%) as compared with control. Neither saline nor furosemide exerted any effect on Na+-K+-ATPase. Moreover, the effect of ouabain alone on Na+-K+-ATPase was not different from that of ouabain plus furosemide. No changes in Mg2+-ATPase were detected in any of the experiments. These results indicate that inhibition of renal Na+-K+-ATPase abolishes furosemide-induced renin secretion despite potentiation of the natriuretic effect of the diuretic. It is apparent that the level of activity of Na+-K+-ATPase is of prime importance for renin secretion. In addition, ouabain may act directly on the juxtaglomerular cells to inhibit renin secretion.     

Effect of thyroid hormone and serum on the development of Na+, K+-adenosine triphosphatase and associated ion fluxes in cultures from rat brain

The effect of culture conditions, serum supplementation or chemically defined medium and the influence of thyroid hormone were studied on the development of the Na+, K+-adenosine triphosphatase (Na+,K+-ATPase) and on the intracellular content of K+ and Na+ ions in cultures which either were greatly enriched in a neuronal cell type, the cerebellar granule cells, or contained a mixed population of cells (brain reaggregates). Foetal rat brain reaggregates displayed lower Na+,K+-ATPase activity when cultured in chemically defined medium than in the presence of serum. Supplementation of the serum-free medium with thyroid hormone resulted in a rise in the Na+,K+-ATPase activity and [3H]ouabain binding to levels similar to those found in the cultures grown in the serum-containing medium. Thyroid hormone had no significant effect on the Mg2+-ATPase activity and on the intracellular content of Na+ and K+ ions. In the granule cell-enriched cerebellar surface cultures the Na+,K+-ATPase activity was lower when the cells were grown in chemically defined medium compared with the serum-containing medium, and the intracellular Na+ to K+ ratio was higher. Thyroid hormone had no effect on the Na+,K+-ATPase activity, [3H]ouabain binding or Mg2+-ATPase activity. The hormone also failed to influence ATPase activities in cerebellar astrocytes maintained in chemically defined medium. Although thyroid hormone had no effect on the Na+,K+-ATPase activity of cultured cerebellar granule cells, treatment with the hormone resulted in a decrease in the ratio of intracellular Na+ to K+ ion content. The effect of the hormone on the Na+,K+-pump activity in live cells was therefore tested by estimating ouabain-sensitive 86Rb uptake. This was regulated as in other cell types, by the rate of Na+ entry: the Na+-ionophore monensin trebled the rate of 86Rb uptake, which was also increased (+30-100%) by 10% foetal calf serum, the maximal response being obtained by about 20 min exposure to serum. The effect was completely blocked by the Na+/H+ exchange inhibitor amiloride. The factor(s) in the serum responsible for the regulation of the Na+,K+-pump were, however, not the thyroid hormones, which failed to affect 86Rb uptake. On the basis of comparing thyroid hormone effects on the different cultures studied it was concluded that not every type of neural cell is target of the hormone action during development.     

A brain Na+, K+-ATPase inhibitor (endobain E) enhances norepinephrine release in rat hypothalamus

We have shown that synaptosomal membrane Na+, K+-ATPase activity is stimulated or inhibited by norepinephrine according to the presence or absence of a brain soluble fraction. Gel filtration of such soluble fraction has allowed the separation of two fractions, peaks I and II, able to stimulate and inhibit Na+, K+-ATPase activity, respectively. Peak II behaves much like ouabain, which has suggested the term endobain. From peak II, a subfraction termed II-E (endobain E), which highly inhibits Na+, K+-ATPase, has been separated by anionic exchange chromatography in a Synchropack AX-300 column. We determined the in vitro effect of endobain E obtained from rat cerebral cortex on neuronal norepinephrine release by incubating rat hypothalamic tissue in the presence of [3H]norepinephrine. Neuronal norepinephrine release was quantified as the factor above basal [3H]norepinephrine released to the medium at experimental and three post-experimental periods. Endobain E was found to increase norepinephrine release in a concentration-dependent fashion, reaching 200%, equivalent to the effect achieved with 400 microM ouabain. Ouabain effect persisted along three post-experimental periods whereas that of endobain E remained only during the first post-experimental period. These results led us to conclude that endobain increases norepinephrine release in hypothalamic neurons at the presynaptic nerve ending level, an effect resembling that of ouabain. It is postulated that endobain E may enhance catecholamine availability in the synaptic gap, leading to an increase in noradrenergic activity.     

Neuronal (Na+,K+)-ATPase and the release of purines from mouse and rat cerebral cortex

Slices of mouse or rat cerebral cortex were incubated with [3H]adenine or [3H]adenosine, and [14C]GABA. Purines and GABA could subsequently be released by ouabain. The release of purines previously shown to occur on restoring elevated K+ levels to normal was not mimicked by noradrenaline at concentrations which activate (Na+,K+)-ATPase. Potassium-free solutions evoked no release of purine during the test period, but resulted in a large release when K+ was restored to normal. K+-free solutions evoked an immediate release of GABA. It is concluded that (Na+,K+)-ATPase is not involved in purine release.     

Inhibition of transcellular NaCl reabsorption in dog kidneys during hypercalcemia

Reduced concentrating and diluting capacity of the kidney in acute and chronic hypercalcemia may partly be due to inhibition of transcellular sodium reabsorption (RNa) in the thick ascending limb of Henle's loop. To examine this hypothesis, local heat production and RNa were measured during normo- and hypercalcemia at comparable glomerular filtration rate (GFR) in volume expanded, anesthetized dogs. Changes in proximal RNa which might occur during CaCl2 infusion, were minimized by infusing acetazolamide (75 mg/kg body wt iv). When ultrafiltrable calcium was increased from 1.12 +/- 0.09 to 2.95 +/- 0.10 mmol/l, cortical heat production was unchanged, whereas outer medullary heat production fell by 32 +/- 4%. RNa was reduced by 32 +/- 6%. Bicarbonate reabsorption did not change but calcium reabsorption and potassium excretion increased significantly. The potassium content of cortex and outer medulla increased during hypercalcemia, whereas ouabain, an inhibitor of Na+, K+-ATPase reduces the potassium content. We conclude that hypercalcemia does not inhibit transcellular RNa in the diluting segment by a direct effect on the Na+, K+-ATPase or the mitochondria, but by interfering with the coupled NaCl transport across the luminal cell membrane.     

Axonal transport of Na+,K+-ATPase identified as a ouabain binding site in rat sciatic nerve

Na+,K+-ATPase levels were measured in different segments of rat sciatic nerves by in vitro binding of [3H]ouabain. Binding sites were found to accumulate on both sides of a ligature tied on the sciatic nerve, indicating an anterograde and retrograde axoplasmic transport of Na+,K+-ATPase. Accumulation of Na+,K+-ATPase at the ligature was time dependent and appeared to occur through fast axoplasmic transport mechanisms. This accumulation on both sides of the ligature was also visualized by autoradiographic studies in longitudinal section of sciatic nerves using [3H]ouabain.     

Effects of magnesium sulfate on Na+,K+ -ATPase and intracranial pressure level after cerebral ischemia.

In the present study, the effects of magnesium sulfate on Na+,K+ -ATPase levels and intracranial pressure (ICP) after cerebral ischemia in rabbits were studied. Thirty New Zealand rabbits were divided into three groups. Group 1 was the control group. In group 2 (untreated group) cerebral ischemia was produced by clamping bilateral common carotid arteries for 60 min but in group 3 magnesium sulfate was administered 100 mg/kg i.v. 10 min after opening the clamps. In group 1, ICP recordings were obtained 5, 60 and 120 min after craniectomy. In groups 2 and 3, ICP recordings were obtained 5 min after craniectomy but before clamping, 60 min after clamping and 60 min after opening the clamps. After taking ICP recordings, brain cortices were resected and Na+,K+ -ATPase activity was determined by subtracting the enzyme activity in the presence of ouabain from the total activity in the absence of ouabain method. There was a significant difference between Na+,K+ -ATPase levels of group 1 and group 2 (P < 0.05). There was no significant difference in Na+,K+ -ATPase levels between group 1 and 3 (P > 0.05), also preischemic ICP values were same in all groups (P > 0.05). Preischemic and postischemic ICP values were significantly different between groups 1 and 2 (P < 0.05), also postischemic (120 min) ICP values were significantly different between group 2 and group 3 (P < 0.05). ICP values correlate well with Na+,K+ -ATPase level. These results demonstrate that cerebral ischemia leads to a decrease of ATPase level in the brain and magnesium sulfate suppresses the decrease of Na+,K+ -ATPase, also magnesium sulfate treatment improves the ICP changes.     

Na+,K+-ATPase enzyme units in lean and obese (ob/ob) thyroxine-injected mice.

The possible involvement of Na+,K+-ATPase in the etiology of obesity in the obese (ob/ob) mouse was explored. The number of Na+,K+-ATPase enzyme units in skeletal muscle, liver, and kidneys from 4- and 8-wk-old obese and lean mice was estimated from saturable [3H]ouabain binding to particulate fractions. Neither phenotype nor age altered the Kd value for ouabain binding in these three tissue preparations. The total number of [3H]ouabain binding sites in hindlimb muscles was 35--55% lower in 4- and 8-wk-old obese mice than in their lean counterparts. However, the total number of [3H]ouabain binding sites in liver and kidneys of obese mice was similar to values observed in their lean counterparts. Because it has been suggested that ob/ob mice are hypothyroid, we investigated the response of Na+,K+-ATPase in these mice to thyroid hormone treatment (approximately 5 microgram thyroxine/day for 2 wk). The number of [3H]ouabain binding sites in the three tissues increased in both obese and lean mice injected with this relatively large dose of thyroxine, but the obese mice were 2--3 times more responsive than lean mice.     

Na+,K+-ATPase activity is inhibited in cultured intestinal epithelial cells by endotoxin or nitric oxide

Na+K+-ATPase is an important enzyme serving vital functions in various mammalian tissues, including the intestine. We have previously documented that endotoxin (LPS) and nitric oxide (NO) can induce enterocyte injury in vitro. To examine whether alterations Na+,K+-ATPase activity might be involved in LPS- or NO-induced enterocyte dysfunction, we carried out four series of experiments. The first set of experiments documented that LPS decreases IEC-6 Na+,K+-ATPase activity at concentrations as low as 0.10 microg/ml. The second set of experiments tested whether exposure of IEC-6 cells to the exogenous NO donor, S-Nitroso-N-acetylpenicillamine (SNAP), would decrease IEC-6 Na+,K+-ATPase activity. The results of these experiments documented that SNAP significantly decreased IEC-6 Na+,K+-ATPase activity in a dose-dependent fashion at a threshold inhibitory concentration of 0.1 mM, and there was an inverse correlation between Na+,K+-ATPase activity and NO concentrations in the medium. Since enterocytes contain iNOS, and LPS can increase iNOS activity, the third set of experiments examined the relationship between LPS-induced inhibition of Na+),K+-ATPase activity and NO production by the IEC-6 cells. These results showed that LPS increased IEC-6 NO production in both a dose- and time-dependent fashion and an inverse correlation existed between LPS-induced NO production and decreased Na+,K+-ATPase activity. Addition of the NOS inhibitor, L-NNA, prevented the LPS-induced decrease in Na+,K+ATPase activity, suggesting that NO is involved in the decrease of Na+,K+-ATPase activity observed in the IEC-6 cells incubated with LPS. One mechanism by which the increased NO concentrations could have contributed to the decrease in Na+,K+ATPase activity, after the addition of LPS or SNAP, is via the production of peroxynitrite during the reaction of NO with superoxide. This notion was supported by studies showing that SNAP- and LPS-induced decreases in IEC-6 Na+,K+-ATPase activity could be blocked by adding superoxide dismutase to the medium. The last set of experiments tested whether the inhibition of Na+,K+-ATPase activity with the specific Na+,K+-ATPase inhibitor ouabain would increase the permeability of an IEC-6 monolayer. IEC-6 monolayer permeability was increased by ouabain, but only at a high concentration. In conclusion, these studies indicate that LPS or the NO donor, SNAP, inhibit Na+,K+-ATPase activity and this inhibition is at least partly related to peroxynitrite production. These studies also suggest that LPS-induced NO production by the IEC-6 cells decreases IEC-6 Na+,K+-ATPase activity in an autocrine fashion.     

The effects of ouabain on resting membrane potential and hyperpolarization-activated current in neonatal rat nodose ganglion neurons

To determine whether the responses of resting membrane potential (RMP) and hyperpolarization-activated current (IH) are altered by the application of ouabain, one of the Na+-K+ pump inhibitors, in neonatal rat small-diameter (<30microm) nodose ganglion (NG) neurons, we examined the effects of 1microM ouabain on those responses using perforated patch-clamp techniques. In current-clamp mode, the RMP was 40.2+/-1.6mV (n=31). Twenty of 31 cells tested were depolarized by ouabain application, and these responses were associated with an increase in the cell input resistance. In the remaining 11 cells studied, 3 showed hyperpolarization in response to ouabain and 8 showed no effect on RMP. In voltage-clamp mode, 1muM ouabain application enhanced the IH in all of 10 neurons examined. These results suggest that ouabain application at 1microM is capable of setting both the RMP level and the neuronal excitability in small-diameter NG neurons.     

Mechanism of NaCl secretion in rectal gland tubules of spiny dogfish (Squalus acanthias)

Rectal gland tubule (RGT) segments of the spiny dogfish (Squalus acanthias) were perfused in vitro. The effects of inhibitors of known mode of action on transepithelial PD (PDte resistance (Rte), the PD across the basolateral membrane (PDbl), the fractional resistance of this membrane (FRbl), and intracellular activities of NA+, Cl-, K+ (apha cell) were examined. Furosemide (5 x 10(-4) mol x 1(-1)) reduced PDte from -12 +/- 0.7 to -2.3 +/- 0.2 mV (n = 63), hyperpolarized PDbl from -71 +/- 1.3 to -79 +/- 0.9 mV (n = 59), FRbl decreased from 0.2 +/- 0.03 to 0.13 +/- 0.01 (n = 21), alpha cell cl- fell from 38 +/- 4 to 11 +/- 2 mmol x 1(-1) (n = 21), alpha cell Na+ fell from 37 +/- 4 to 17 +/- 2 mmol x 1(-1) (n = 12) and alpha cell K+ was constant [113 +/- 14 vs. 117 +/- 15 mmol x 1(-1) (n = 6)]. Furosemide exerted its effects within some 20-40s. Its action was completely reversible. Analysis of the time courses revealed that the furosemide induced initial fall in alpha cell cl- was approximately twice as rapid when compared to that of alpha cell Na+. Ba2+ 0.5 mmol x 1(-1) (bath) reduced PDte from -7.1 +/- 1.2 to -4.1 +/- 0.6 mV (n = 24), increased Rte from 18 +/- 2 to 22 +/- 2.5, omega cm2 (n = 14). PDbl depolarized from -75 +/- 2 to -48 +/- 2 mV (n = 42), FRbl increased from 0.2 +/- 0.02 to 0.34 +/- 0.04 (n = 14) and alpha cell K+ increased from 143 +/-28 to 188 +/- mmol x 1(-1) (n = 4). Ouabain (50 x 10(-6) mol x 1(-1), bath) reduced PDte from -12 +/-2 to -3 +/- 0.5 mV (n = 9), Rte increased from 18 +/- 3 to 21 +/- 3 omega cm2 (n = 5). PDbl depolarized from -67 +/- 4 to -26 + 3 mV (n = 14), FRbl increased from 0.23 +/- 0.04 to 0.45 +/- 0.05 (n = 6), alpha cell K+ fell only slightly from 135 +/- 15 to 112 +/- 30 mmol x 1(-1) (n = 4), but alpha cell cl- increased from 35 +/- 12 to 111 +/- 37 mmol x 1(-1) (n = 3). These effects of ouabain were slow when compared to those exerted by furosemide or Ba2+. The ouabain effects on PDte and PDbl were completely prevented if furosemide was applied first.(ABSTRACT TRUNCATED AT 400 WORDS)     

Catecholamine release from bovine chromaffin cells: the role of sodium-calcium exchange in ouabain-evoked release

Spontaneous catecholamine (CA) release from bovine chromaffin cells maintained in primary tissue culture has been measured after pre-loading the cells with [3H]noradrenaline. Ouabain inhibited 86Rb+ uptake and increased 3H release in a concentration-dependent manner during a 60 min incubation period. Low external Na+ (5 mM: Li+ substitution) also increased 3H release. Whereas the 3H-releasing action of ouabain was maintained, the Li(+)-evoked release decreased with time. The effects of both ouabain and low Na+ solution on 3H release were completely inhibited by removal of Ca2+ from the external medium even though in Ca2(+)-free solution ouabain further inhibited 86Rb+ uptake into the cells. Readmission of Ca2+ to Na(+)-loaded cells (10-4 M-ouabain in Ca2(+)-free-1 mM-EGTA solution for 60 min) markedly increased the release of 3H. In the additional presence of diphenylhydantoin (DPH, 10-4 M) 3H release was significantly less on Ca2+ readmission. The 3H release from Na(+)-loaded cells was proportional to the concentration of Ca2+ readmitted. The 3H release was further increased from Na(+)-loaded cells in response to Ca2+ readmission when [Na+]o was lowered from 149 to 5 mM (Li+, choline+, Tris+ or sucrose substitution) though Li+ was less effective than the other Na+ substitutes. Potassium removal from the external medium significantly inhibited the 3H release evoked by Ca2+ readmission to Na(+)-loaded cells, even when [Ca2+]o was greater than normal (7.5 mM) or if Ca2+ was readmitted in low [Na+]o solution. Rb+, Cs+ or Li+ could substitute for K+ with the order of potency: Rb+ greater than or equal to K+ greater than Cs+ greater than Li+. A slight increase of external K+ (10.8 mM) potentiated the 3H release from Na(+)-loaded cells on Ca2+ readmission, but a higher concentration of K+ (149.4 mM) had the opposite action. The data is consistent with the hypothesis that ouabain-evoked CA release from bovine chromaffin cells is, in part, a consequence of an internal Na(+)-dependent Ca2+ influx. The evidence also suggests that there is Na(+)-Ca2+ competition at the external arm of the exchanger together with a monovalent cation activation site.     

Two different modes of inhibition of the rat brain Na+, K(+)-ATPase by triterpene glycosides, psolusosides A and B from the holothurian Psolus fabricii

Effects of two triterpene glycosides, isolated from the holothurian Psolus fabricii, on rat brain Na+, K(+)-ATPase (Na, K-pump; EC 3.6.1.3) were investigated. Psolusosides A and B (PsA and PsB) inhibited rat brain Na+, K(+)-ATPase with I50 values of 1 x 10(-4) M and 3 x 10(-4) M, respectively. PsA significantly stimulated [3H]ATP binding to Na+, K(+)-ATPase, weakly increased [3H]ouabain binding to the enzyme, and inhibited K(+)-phosphatase activity to a smaller degree than the total reaction of ATP hydrolysis. In contrast, PsB decreased [3H]ATP binding to Na+, K(+)-ATPase, and had no effect on [3H]ouabain binding to the enzyme. K(+)-Phosphatase activity was inhibited by PsB in parallel with Na+, K(+)-ATPase activity. The fluorescence intensity of tryptophanyl residues of Na+, K(+)-ATPase was increased by PsA and decreased by PsB in a dose-dependent manner. The excimer formation of pyrene, a hydrophobic fluorescent probe, was decreased by PsA only. The different characteristics of inhibition mode for these substances were explained by peculiarities of their chemical structures and distinctive affinity to membrane cholesterol.     

Permeation of NH3/NH4 + and cell pH in colonic crypts of the rat

Colon cells are subjected to high concentrations of NH3 and NH4+, and a sizeable portion of this buffer is absorbed. The flux of these components into cells causes opposite effects on their pH; this effect is largely used to induce an acid load and to observe the mechanism of acid extrusion from cells. We studied cells of microdissected colon crypts loaded with BCECF and superfused with NH4Cl-containing Krebs-Ringer solution. We found a marked transient reduction in pH measured by ratiometric fluorescence microscopy, from a control value of 7.51 +/- 0.041 to 7.15 +/- 0.041 (n = 21), instead of the initial alkalinization found in most cells. This pH was reached at a rate of 0.95 +/- 0.07 pH units/min. Addition of 1 mmol/l furosemide, a blocker of Na+,K+,2Cl- cotransport, to the ammonium solution inverted this acidification toward alkalinization (pH 7.89 +/- 0.041, n = 5), and superfusion with furosemide plus 0.1 mmol/l hexamethylene amiloride, a specific blocker of Na+/H+ exchange, increased this initial alkalinization further to 8.10 +/- 0.117 (n = 7). When Krebs-Ringer with 0 Cl- containing (NH4)2SO4 instead of NH4Cl was superfused, the acid transient was also reverted to alkalinization; however, a higher degree of alkalinization was observed either when 1 mmol/l furosemide was added to the superfusing sulfate solution (when a pH of 7.78 +/- 0.010 was reached), or when ammonium gluconate was used instead of ammonium sulfate. The addition of Ba2+ to the superfusion solution did not alter the initial acidification. These data indicate that in colon crypt cells, basolateral membrane transporters, in particular the Na+,K+,2Cl- cotransporter and the Na+/H+ exchanger (but not Ba(2+)-sensitive K+ channels), mediate the predominant influx of NH4+ ions leading to the initial transient acidification.     

Mammalian ion-coupled solute transporters.

Active transport of solutes into and out of cells proceeds via specialized transporters that utilize diverse energy-coupling mechanisms. Ion-coupled transporters link uphill solute transport to downhill electrochemical ion gradients. In mammals, these transporters are coupled to the co-transport of H+, Na+, Cl- and/or to the countertransport of K+ or OH-. By contrast, ATP-dependent transporters are directly energized by the hydrolysis of ATP. The development of expression cloning approaches to select cDNA clones solely based on their capacity to induce transport function in Xenopus oocytes has led to the cloning of several ion-coupled transporter cDNAs and revealed new insights into structural designs, energy-coupling mechanisms and physiological relevance of the transporter proteins. Different types of mammalian ion-coupled transporters are illustrated by discussing transporters isolated in our own laboratory such as the Na+/glucose co-transporters SGLT1 and SGLT2, the H(+)-coupled oligopeptide transporters PepT1 and PepT2, and the Na(+)- and K(+)-dependent neuronal and epithelial high affinity glutamate transporter EAAC1. Most mammalian ion-coupled organic solute transporters studied so far can be grouped into the following transporter families: (1) the predominantly Na(+)-coupled transporter family which includes the Na+/glucose co-transporters SGLT1, SGLT2, SGLT3 (SAAT-pSGLT2) and the inositol transporter SMIT, (2) the Na(+)- and Cl(-)-coupled transporter family which includes the neurotransmitter transporters of gamma-amino-butyric acid (GABA), serotonin, dopamine, norepinephrine, glycine and proline as well as transporters of beta-amino acids, (3) the Na(+)- and K(+)-dependent glutamate/neurotransmitter family which includes the high affinity glutamate transporters EAAC1, GLT-1, GLAST, EAAT4 and the neutral amino acid transporters ASCT1 and SATT1 reminiscent of system ASC and (4) the H(+)-coupled oligopeptide transporter family which includes the intestinal H(+)-dependent oligopeptide transporter PepT1.     

Characterization of a Na+-dependent betaine transporter with Cl- channel properties in squid motor neurons

Most marine invertebrates, including squids, use transporters to accumulate organic osmolytes such as betaine, to prevent water loss when exposed to elevated salinity. Although a limited number of flux studies have shown the Na+ dependence of betaine transport, nothing is known about the electrogenic properties of osmolyte transporters. We used whole cell and perforated-patch voltage-clamp techniques to characterize the electrical properties of the betaine transporter in giant fiber lobe motor neurons of the squid Lolliguncula brevis. Betaine activated a large, Cl--selective current that was reversibly blocked by 100 microM niflumic acid (97 +/- 2% block after 40 s, SD; n = 7) and partially inhibited by 500 microM SITS (29 +/- 11%; n = 5). The Cl- current was Na+ dependent and was virtually eliminated by isotonic replacement of Na+ with Li+, NMDG+, or Tris+. Concentration-response data revealed an EC50 in a physiologically relevant range for these animals of 5.1 +/- 0.9 mM (n = 11). In vertebrates, the betaine transporter is structurally related to the GABA transporter, and although GABA did not directly activate the betaine-induced current, it reversibly reduced betaine responses by 34 +/- 14% (n = 8). Short-term changes in osmolality alone did not activate the Cl- current, but when combined with betaine, Cl- currents increased in hypertonic solutions and decreased in hypotonic solutions. Activation of the betaine transporter and Cl- current in hypertonic conditions may affect both volume regulation and excitability in L. brevis motor neurons. This study is the first report of a novel betaine transporter in neurons that can act as a Cl- channel.     

Membrane excitability, weakness, and fatigue.

A failure in membrane excitability, defined as an inability of the sarcolemma and T-tubule to translate the neural discharge command into repetitive action potentials, represents an inviting cause of mechanical disfunction in both health and disease. A failure at this level would precipitate a disturbance in signal transmission between the T-tubule and the calcium release channels of the sarcoplasmic reticulum, resulting in reduced release of Ca2+, lower cytosolic free Ca2+ levels, and depressed myofibrillar activation and force generation. The ability of the sarcolemma and T-tubules to conduct repetitive action potentials is intimately dependent on active transport of Na+ and K+ following an action potential. The active transport of these cations is mediated by the Na+-K+-ATPase, an integral membrane protein that uses the energy from the hydrolysis of 1 ATP to transport 3 Na+ out of the cell and 2 K+ into the cell. A failure to recruit sufficient Na+-K+-ATPase activity during contractile activity could result in a rundown of the transmembrane gradients for Na+ and K+, leading to a loss of membrane excitability. The Na+-K+-ATPase activity depends on the amount and isoform composition of the protein, substrate availability, and acute regulatory factors. Each of these factors is examined as a potential cause of altered activation of the Na+-K+-ATPase activity and loss of membrane excitability in fatigue. Regular exercise represents a potent stimulus for upregulating Na+-K+-ATPase levels and for increasing the ability for cation transport across the sarcolemma and T-tubule membrane. As such, training may be a valuable tool in the management of fatigue in health and disease.