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.     

Inhibition of hyphal growth of Candida albicans by activated lansoprazole, a novel benzimidazole proton pump inhibitor.

The effect of activated lansoprazole (AG 2000), a novel benzimidazole proton pump inhibitor, against hypha formation of Candida albicans was examined in hypha-forming medium pH 7 (HFM7) after 20 h. AG 2000, at 50-800 microM, did not inhibit germ tube formation. However, it inhibited elongation of germ tubes to form hyphae and favored conversion of germ tubes to resume yeast growth at concentrations of > or =200 microM. Pre-treatment of AG 2000 with a sulfhydryl reagent (1:1), such as 2-mercaptoethanol. blocked the inhibitory property of AG 2000 on hypha formation.     

Intracellular sodium modulates the state of protein kinase C phosphorylation of rat proximal tubule Na+,K+-ATPase

The natriuretic hormone dopamine and the antinatriuretic hormone noradrenaline, acting on alpha-adrenergic receptors, have been shown to bidirectionally modulate the activity of renal tubular Na+,K+-adenosine triphosphate (ATPase). Here we have examined whether intracellular sodium concentration influences the effects of these bidirectional forces on the state of phosphorylation of Na+,K+-ATPase. Proximal tubules dissected from rat kidney were incubated with dopamine or the alpha-adrenergic agonist, oxymetazoline, and transiently permeabilized in a medium where sodium concentration ranged between 5 and 70 mM. The variations of sodium concentration in the medium had a proportional effect on intracellular sodium. Dopamine and protein kinase C (PKC) phosphorylate the catalytic subunit of rat Na+,K+-ATPase on the Ser23 residue. The level of PKC induced Na+,K+-ATPase phosphorylation was determined using an antibody that only recognizes Na+,K+-ATPase, which is not phosphorylated on its PKC site. Under basal conditions Na+,K+-ATPase was predominantly in its phosphorylated state. When intracellular sodium was increased, Na+,K+-ATPase was predominantly in its dephosphorylated state. Phosphorylation of Na+,K+-ATPase by dopamine was most pronounced when intracellular sodium was high, and dephosphorylation by oxymetazoline was most pronounced when intracellular sodium was low. The oxymetazoline effect was mimicked by the calcium ionophore A23187. An inhibitor of the calcium-dependent protein phosphatase, calcineurin, increased the state of Na+,K+-ATPase phosphorylation. The results imply that phosphorylation of renal Na+,K+-ATPase activity is modulated by the level of intracellular sodium and that this effect involves PKC and calcium signalling pathways. The findings may have implication for the regulation of salt excretion and sodium homeostasis.     

The effect of opioids and of naloxone on Na+,K+-adenosine triphosphatase activity in frog spinal cord membrane fractions

The effects of opioids and of naloxone on ouabain-sensitive Na+,K+-adenosine triphosphatase (ATPase) activity were studied in vitro on membrane fractions from frog spinal cords. The addition of morphine and of the stable enkephalin analogue, D-Ala2,D-Leu5-enkephalin, in concentrations from 10(-7) to 10(-4) M significantly increased Na+,K+-ATPase activity. No effect was found with methionine enkephalin (Met-Enk). However, the addition of two peptidase inhibitors, captopril and phosphoramidon (10(-5) M each), significantly increased Na+,K+-ATPase activity. A further increase in enzyme activity was found when Met-Enk (10(-4) or 10(-7) M) was added simultaneously with peptidase inhibitors. On the other hand, the addition of the opiate antagonist, naloxone, at low concentration (10(-7) M) decreased the activity of Na+,K+-ATPase. These results are discussed with respect to the effect of synthetic and endogenous opioids on the activity of Na+,K+-ATPase.     

The presumptive identification of Candida albicans with germ tube induced by high temperature

For direct identification of Candida albicans from other Candida species, the chlamydospore formation and the mycelial transition induced by high temperature and by sera were examined in 198 Candida isolates. The germ tubes of C. albicans developed early at 30 min in high temperature-induction, but at 60 min in serum-induction. C. albicans generated germ tubes well at concentrations lower than 2 x 10(7) cells/ml, but the germ tube formation was markedly restrained at concentrations higher than 4 x 10(7) cells/ml. In a serum-free, yeast extract-peptone-dextrose (YEPD) medium, C. albicans grew as a yeast form at 30 degrees C and as a mycelial form at 35-42 degrees C. Mycelial development was maximal at 37 degrees C in serum and at 39 degrees C in YEPD. Germ tubes were formed within 30 min in YEPD at 39 degrees C, but after 60 min in serum at 37 degrees C. Our examination showed that the 39 degrees C-induced germ tube formation tests were very reliable (sensitivity 100%, specificity 100%) at discerning C. albicans from other Candida species. These results suggest that the high temperature-induced germ tube formation testing could be a useful identification method of C. albicans in clinical laboratories.     

Modulation of force development by Na+, K+, Na+ K+ pump and KATP channel during muscular activity.

Extracellular K+ concentration increases during exercise and especially during fatigue development. It has been proposed that K+ is an important factor in the etiology of skeletal muscle fatigue because it suppresses membrane excitability and eventually force development. Based on the effect of K+, it has then been proposed the Na+ K+ pump reduces increases in extracellular K+ concentration while the ATP-sensitive K+ channel (KATP channel) allows for rapid increases in extracellular K+ to suppress force development when ATP levels start to fall or when the levels of metabolic end-products become high. However, recent studies have now demonstrated that an increase in extracellular K+ concentration can be advantageous to muscle during exercise because it not only stimulates vasodilation and the exercise pressor reflex, but it also potentiates force development when the Na+ concentration gradient is maintained. A new hypothesis is therefore proposed in which the Na+ K+ pump is important in maintaining the Na+ concentration gradient (and not the K+ concentration gradient as previously suggested), while the activation of KATP channels is important to increase the K+ efflux and extracellular concentration. This situation then optimizes the development of force during exercise. Another hypothesis is proposed in which more KATP channels are activated while the activity of the Na+ K+ pump is reduced when ATP levels start to decrease to allow for an accumulation of intracellular Na+ and further increases in extracellular K+ concentration. These concentration changes then reduce membrane excitability and force development (i.e., fatigue) to protect muscle against large ATP depletion and function impairment.     

Potential dependency of the electrogenic Na+-pump current in bullfrog atrial muscles

Experiments were designed to evaluate the concept that the activity of the electrogenic Na+-pump is dependent on the transmembrane potential. Cardiac muscle preparations were used because the electrogenic Na+-pump current can be recorded at different potentials with the voltage clamp method in this preparation. Electrogenic Na+-pump current was identified as the membrane current which was abolished by ouabain (5 microM) and induced by the addition of K+ or an alkali metal cation, such as Rb+, Cs+, or Li+, to the extracellular K+-free solution. Alkali metal cations other than K+ were used to eliminate the possibility that a passive membrane K+ current might be altered by changes in the K+ concentration in the vicinity of the membrane due to activation of the Na+-pump. It was concluded that the activity of the electrogenic Na+-pump current is dependent on the membrane potential.     

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.     

External K+ increases Na+ conductance of the hyperpolarization-activated current in rabbit cardiac pacemaker cells.

The hyperpolarization-activated current (I(f)) was recorded from single myocytes dissociated from rabbit sinoatrial node. Although I(f) is usually carried by both Na+ and K+, removal of the minor K+ component from physiological saline suppresses inward component. This inward Na+ current through I(f) channel increases on raising the extracellular K+ concentration. The Na+ conductance relative to K+ conductance (PNa/PK), as measured from the reversal potential, increases and saturates near 5 mM K+. This effect is different from the current increase caused by raising the concentration of carrier ion K+, which saturates at 70 mM with a half-maximal value (K1/2) of 10 mM. It is suggested that the I(f) channel has multiple, interactive binding sites for cation permeation.     

Regulation of K+ transport in the rat distal colon via angiotensin II subtype receptors and K+ -pathways

The role of angiotensin subtype-1 (AT1) and -2 (AT2) receptors in mediating the effects of angiotensin II (ANG II) on several K+ transporters was studied in rat distal colon using an Ussing chamber. Angiotensin II induced K+ secretion at two different doses. Secretion occurred at 10-(8) and 10-(4) M, as a result of an increase in serosal-to-mucosal flux (Js-m). The ANG II-induced stimulation of Js-m at a low dose (10-(8) M) was abolished by PD123319 while losartan did not alter the low-dose ANG II-dependent increase in Js-m. In contrast, the increase in Js-m induced by a high-dose of ANG II (10-(4) M) was blocked by losartan, whereas PD123319 partially reduced the stimulatory effect. In the presence of both blockers, high-dose ANG II induced an inhibition of basal Js-m. Low-dose ANG II activated the barium-sensitive K+ channels, whereas the Na+, K+, 2Cl- cotransporter and the Na+, K+ -ATPase pump were unchanged. At the high dose, ANG II activated the barium-sensitive K+ channels and the Na+, K+, 2Cl- cotransporter and inhibited the Na+, K+ -ATPase pump. These data indicate that ANG II stimulates serosal-to-mucosal K+ flux in the rat distal colon at high and low doses via different receptors and K+ transporters.     

Barium-induced inhibition of K+ transport mechanisms in cortical astrocytes--its possible contribution to the large Ba2+-evoked extracellular K+ signal in brain

Homogenous mouse astrocytes in primary cultures were used to investigate the action of different Ba2+ concentrations on 42K transport, membrane potential and Na+,K+-adenosine triphosphatase activity. Five millimolar Ba2+ reduced total K+ influx and efflux (each by 83%) and ouabain-sensitive net K+ uptake (by 80%); it decreased the K+ content, depolarized the membrane potential reversibly and completely inhibited the Na+,K+-adenosine triphosphatase activity. The concentration dependence of these effects was biphasic. Concentrations between 2 and 20 microM affected only the passive K+ fluxes (IC50: 6 microM). Concentrations between 50 microM and 5 mM inhibited the Na+,K+-adenosine triphosphatase and had no further effect on passive fluxes, but inhibited the ouabain-sensitive net uptake of K+ (IC50: 3.1-0.6 mM). It is suggested that the large evoked extracellular K+ increase in the brain observed in Ba2+-treated preparations in vivo or in brain slices to a large extent is due to the impairment of passive and active K+ clearance by glial cells.     

Candidacidal activities of proteins partially purified from rat epidermis.

Proteins with approximate molecular weights of greater than 300,000 (EP greater than 300K) and 49,000 (EP 49K) were partially purified from terminally differentiated cells of 2-day-old rat epidermis. They were extracted in 0.34 M sucrose containing 0.01 M citric acid and purified by Sephacryl S-300 chromatography followed by reverse-phase column chromatography. The major constituents of EP greater than 300K and EP 49K were focused around pH 10 to 11 by sucrose gradient isoelectric focusing. Both proteins were effective at inhibiting colony formation of Candida albicans and C. tropicalis, but neither inhibited the growth of C. parapsilosis. The effect was maximum below pH 5.0 and reduced considerably above pH 5.0. The activity of EP greater than 300K on C. albicans TIMM 1623 (group A) was much stronger than that of EP 49K, whereas both proteins similarly inhibited C. albicans TIMM 1604 (group B). Their effects against C. albicans TIMM 1623 were dose dependent and were activated after a longer preincubation time, and NaCl concentration influenced their potency. At a low salt concentration and a 60-min preincubation at pH 4.5, the 50% effective dose for EP greater than 300K was calculated to be 1.7 x 10(-9) M, whereas that for EP 49K was 1.8 x 10(-7) M.     

Requirement of calcium ions for cell degeneration with a toxin (vibriolysin) from Vibrio parahaemolyticus.

A highly purified toxin (vibriolysin) from Vibrio parahaemolyticus caused degeneration of cell shape, such as bleb and balloon formation, of mouse myocardial cells and mouse melanoma cells in culture. An extracellular Ca2+ concentration of more than 10(-6) M was necessary for the degeneration of cell shape, but extracellular Mg2+, Na+, and K+ were not necessary. In the presence of extracellular Ca2+, vibriolysin also caused full contraction of myofibrils of mouse myocardial cells and reduction of both actin cables and tubulin networks of mouse melanoma cells. Vibriolysin also caused excess uptake of Ca2+ from the incubation medium by mouse myocardial cells and mouse melanoma cells. Chick myocardial cells, which show neither degeneration of cell shape nor full contraction of myofibrils, did not take up excess 45Ca2+ in the presence of vibriolysin. These findings suggest that the vibriolysin-induced degeneration of cell shape of mouse myocardial cells and mouse melanoma cells is due to excess uptake of Ca2+ from the incubation medium by the cells.     

Inductions of germ tube and hyphal formations are controlled by mRNA synthesis inhibitor in Candida albicans.

Candida albicans is a pathogenic dimorphic fungus. When yeast cells were pre-incubated in YPD medium at 25degreesC and released into HFM7 medium containing 4% serum at 37degreesC, germ tubes emerged within 0.5 h. To determine whether mRNA or protein synthesis was necessary for germ tube formation, we examined the effects of mRNA and protein syntheses inhibitors on this formation. In the presence of cycloheximide, cells were unbudded and no germ tube was observed. However, in the presence of actinomycin D, germ tube formation was observed while budding growth and true hyphae elongation were blocked. Next, we measured mRNA or protein accumulation during induction of germ tube formation in the presence of the inhibitors. In the presence of cycloheximide, protein was not synthesized, while in the presence of actinomycin D, mRNA synthesis decreased to 6.3% and protein synthesis to 37.7%. The condition we found which allows only germination but not budding or filamentation might be convenient to use in screening genes involved in the initial stage of morphological change in C. albicans.     

Recovery of intracellular pH in cortical brain slices following anoxia studied by nuclear magnetic resonance spectroscopy: role of lactate removal, extracellular sodium and sodium/hydrogen exchange.

[31P]- and [1H]nuclear magnetic resonances recorded in an interleaved fashion were used in order to quantify high-energy phosphates, intracellular pH and lactate in cortical brain slices of the guinea-pig superfused in a CO2/HCO3(-)-buffered medium during and after anoxic insults. The volume-averaged intracellular pH and energy status of the preparation following anoxia were determined. In the presence of external Na+, intracellular pH normalized in 3 min and was significantly more alkaline from 10 to 12 min of recovery, but lactate remained elevated for 12 min of reoxygenation following anoxia. The amount of lactate removed was only 40% of the quantity of acid extruded showing operation of H+ neutralizing transmembrane mechanisms other than transport of lactic acid. Amiloride (1 or 2 mM) did not prevent the recovery of intracellular pH, but it blocked the "overshoot" of the alkalinization at 10-12 min of recovery. In a medium containing 70 mM K+, 60 mM Na+ and 0.1 mM Ca2+, the recovery of pH, but not lactate washout, was significantly delayed. Removal of external Na+ caused severe energetic failure, decreases both in oxygen uptake and in N-acetyl aspartate concentration, indicating loss of viable tissue. In Na(+)-free superfusion, lactic acidosis caused a more severe drop in intracellular pH than in the presence of Na+. Complexing of extracellular Ca2+ in the Na(+)-free medium inhibited the acidification by 0.38 pH units during anoxia which is as much as the acidification caused by lactate accumulation in the absence of Na+. In Na(+)-free medium intracellular pH recovered, however, from an anoxic level to a normoxic value in 6 min. Metabolic damage of the slice preparation induced by anoxia in the absence of Na+ was as profound in the presence as in the absence of Ca2+ showing that accumulation of Ca2+ is not the only reason for the damage. It is concluded that recovery of intracellular pH from lactic-acidosis can occur independently of energetic recovery and involves acid extrusion mechanism(s) that is(are) dependent on external Na+ and sensitive to high K+.     

Effect of insulin upon membrane-bound (Na+ + K+)-ATPase extracted from frog skeletal muscle.

1. Insulin stimulates the activity of membrane-bound ATPase isolated from frog skeletal muscle and from rat brain. The increase in activity of the membrane-bound ATPase system isolated from frog ranged from 9-8 to 53% at concentrations of Na+ (25 mM), K+ (10 mM), and ATP (2 mM) similar to those in in vivo experiments conducted previously (Moore, 1973). The increased activity of the membrane-bound ATPase is, therefore, at least as great as the insulin-induced increase in Na efflux (10-38%) from intact cells (Moore, 1973). If the concentration of Na+ is lowered to 4 mM and that of ATP lowered to 0-5 mM albumin, and 10(6) M, the increase in ouabain-inhibitable ATPase activity can reach as high as 400%. 2. Ouabain, at a concentration (10(-3) M) sufficient to inhibit stimulation of the frog ATPase by increasing Na from 4 to 25 mM, completely blocked the stimulation of ATPase activity due to insulin. 3. At 2 mM-ATP, 100 mM-Na+, and 20 mM-K+, conditions which maximally activate the (Na+ + K+)-ATPase, insulin did not increase the ATPase, activity. Stimulation was consistently seen at 10 mM-K+, 0-5 mM-ATP, and either 4 mM or 25 mM-Na+. 4. The finding that insulin does not stimulate the ATPase activity in conditions in which the (Na+ + K+)-ATPase component is maximally activated and especially the fact that ouabain can reproducibly inhibit insulin stimulation of the membrane-bound ATPase activity strongly suggest that interaction of insulin with its receptor upon the plasma membrane somehow stimulates the (Na+ + K+)-ATPase system (ouabain sensitive; ATP phosphohydrolase, EC (3.6.1.3). These results are consistent with previous studies of the effect of insulin upon Na efflux from intact cells (Moore, 1973) and support the previous conclusion that the component of Na efflux stimulated by insulin is active. The evidence suggests that insulin probably does not affect Vmax of the (Na+ + K+)-ATPase system, but may increase the affinity of the enzyme system to one or more effectors, most likely Na+, ATP, and perhaps K+. 5. Oxidized glutathione (2-7 X 10(-6) M), 10(-6) M, 10(-7) M, and 10(-8) M cyclic AMP did not affect the ATPase activity 10(-6)Malbumin, and . 6. The results are consistent with the view that the Na pump, (Na+ + K+)-ATPase, is intimately involved with the physiological action of insulin and may be transducer between the binding of insulin to its receptor on the plasma membrane and the cellular actions of insulin.     

Avian shell gland contractility: interaction of PGF2 alpha and arginine vasotocin with Ca2+

The effects of prostaglandin F2 alpha (PGF2 alpha) and arginine vasotocin (AVT) on the isometric contractile activity of avian shell gland longitudinal muscle strips were studied in relation to the role of extracellular Ca2+. PGF2 alpha and AVT stimulated contractile tension in a dose-related manner. This was attenuated when either of the Ca2+ channel blockers verapamil or R33956 was added to the muscle chamber baths. Ca2+-free solution containing 1 mM ethyleneglycol-bis (beta-aminoethylether)-N,N'-tetraacetic acid completely prevented contraction in response to increasing doses of PGF2 alpha or AVT. Washing of the strips with Ca2+-free solution eliminated spontaneous contractile activity, but replenishment of CaCl2 to the medium (0.1-5.0 mM) restored it. Addition of PGF2 alpha to the Ca2+-free medium enhanced contractile tension during Ca2+ replenishment, whereas AVT had no effect on tension generation at low extracellular Ca2+ concentration (0.1-0.5 mM) but increased it at higher extracellular Ca2+ concentration (1.0-5.0 mM). PGF2 alpha stimulation was sensitive to extracellular Na+ concentration, whereas AVT-induced activity was not. Potassium depolarization (20 mM K) potentiated PGF2 alpha-stimulated activity, whereas the response to AVT was unaffected. At 127.5 mM K, AVT-stimulated activity was inhibited. PGF2 alpha-enhanced Ca2+-dependent tension generation was right shifted in a dose-related manner by AVT. These results suggest that extracellular Ca2+ is necessary for the full expression of PGF2 alpha- and AVT-stimulated muscle contraction and suggest that each agonist has a different mechanism of action.     

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.     

Extracellular hydrogen ions influence channel-mediated and carrier-mediated K+ fluxes in cultured mouse astrocytes

Physiological (pH 7.2 and 7.0) and pathological (pH 6.8 and 6.6) changes of external pH, as they are measured in vivo, were imposed on mouse astrocytes in primary cultures and the effect on components of K+ transport pathways across the cell membrane was measured. Physiological pH changes had no effect at all. Pathological pH changes in inhibited K+ fluxes through channels by 40%. This effect occurred 3-6 min after a pH change and was not additive to a similar change induced by amiloride. This could be seen as an indication that the observed effects are mediated by subsequent changes in intracellular pH; however, direct intracellular pH measurements were not undertaken. A low pH of 6.6 inhibited the activity of the Na+, K+-ATPase by 65% and reduced the carrier-mediated K+ net accumulation into the cells by 60%. This suggests that measured extracellular pH changes in the brain may be functionally important, as they interfere with the K+ homeostasis of the nervous tissue.     

Cation effects on acid secretion in rabbit gastric glands

The ability of isolated gastric glands to produce acid as a function of intra- and extracellular concentrations of K+ and Na+ was investigated. Ouabain inhibited acid formation as measured by the aminopyrine (AP) accumulation technique in a dose-dependent manner with an ED50 of 2 X 10(-6) M at 5.4 mM extracellular K+. This inhibition was counteracted by increasing extracellular K+ or decreasing extracellular Na+. In K+-free solutions with regular Na+, the AP accumulation was almost totally abolished; the readdition of K+ rapidly restored the response with a K0.5 of 1 mM extracellular K+. In the absence of Na+, with or without ouabain, there was always a significant residual AP accumulation, but the K0.5 of extracellular K+ required for acid formation increased to about 20 mM. Despite repeated washings in Na+-K+-free solutions, the intracellular K+ content could not be decreased below 24 mM above the apparent K0.5 for K+. It was found that the intracellular K+ could be depleted without disturbance of the acid secretory function if the glands were treated with the neutral ionophore amphotericin B and ouabain in K+-Na+-free solutions. Complete dose-response curves of H+ secretion as a function of K+ concentration could now be obtained. Thus the K0.5 for K+ activation of AP accumulation was 16.5 +/- 0.9 mM; upon histamine stimulation of the glands, the K0.5 decreased to 10.4 +/- 0.7 mM. Intracellular K+ concentrations above approximately 60 mM inhibited AP accumulation. Na+ dose dependently inhibited the K+-induced response. At low extracellular K+ concentrations, a ouabain-insensitive K+ accumulation mechanism was unmasked. This K+ uptake was partly inhibited y p-chloromercuribenzene sulfonic acid and by inhibitors of the gastric H+-K+-ATPase and amplified by inhibitors of K+ conductance channels such as Ba2+. The K+ content of gastric glands is mainly regulated by the Na+-K+-ATPase, but additional K+-uptake pathways are present. The affinity for K+ is increased in histamine-stimulated glands, which might indicate one mechanism of stimulating the secretory response. Na+ inhibits secretion possibly by binding to the cytosolic cation site of the gastric ATPase. The cation relationship to acid secretion in the rabbit parietal cell is similar to that described for vesicles isolated from hog gastric mucosa.