Na+, K+, and Cl- transport in resting pancreatic acinar cells

To understand the role of Na+, K+, and Cl- transporters in fluid and electrolyte secretion by pancreatic acinar cells, we studied the relationship between them in resting and stimulated cells. Measurements of [Cl-]i in resting cells showed that in HCO3(-)-buffered medium [Cl- ]i and Cl- fluxes are dominated by the Cl-/HCO3- exchanger. In the absence of HCO3-, [Cl-]i is regulated by NaCl and NaK2Cl cotransport systems. Measurements of [Na+]i showed that the Na(+)-coupled Cl- transporters contributed to the regulation of [Na+]i, but the major Na+ influx pathway in resting pancreatic acinar cells is the Na+/H+ exchanger. 86Rb influx measurements revealed that > 95% of K+ influx is mediated by the Na+ pump and the NaK2Cl cotransporter. In resting cells, the two transporters appear to be coupled through [K+]i in that inhibition of either transporter had small effect on 86Rb uptake, but inhibition of both transporters largely prevented 86Rb uptake. Another form of coupling occurs between the Na+ influx transporters and the Na+ pump. Thus, inhibition of NaK2Cl cotransport increased Na+ influx by the Na+/H+ exchanger to fuel the Na+ pump. Similarly, inhibition of Na+/H+ exchange increased the activity of the NaK2Cl cotransporter. The combined measurements of [Na+]i and 86Rb influx indicate that the Na+/H+ exchanger contributes twice more than the NaK2Cl cotransporter and three times more than the NaCl cotransporter and a tetraethylammonium-sensitive channel to Na+ influx in resting cells. These findings were used to develop a model for the relationship between the transporters in resting pancreatic acinar cells.     

Na+-K+ pump activation inhibits endothelium-dependent relaxation by activating the forward mode of Na+/Ca2+ exchanger in mouse aorta

The effect of Na+-K+ pump activation on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) was examined in mouse aorta and mouse aortic endothelial cells (MAECs). The Na+-K+ pump was activated by increasing extracellular K+ concentration ([K+]o) from 6 to 12 mM. In aortic rings, the Na+ ionophore monensin evoked EDR, and this EDR was inhibited by the Na+/Ca2+ exchanger (NCX; reverse mode) inhibitor KB-R7943. Monensin-induced Na+ loading or extracellular Na+ depletion (Na+ replaced by Li+) increased [Ca2+]i in MAECs, and this increase was inhibited by KB-R7943. Na+-K+ pump activation inhibited EDR and [Ca2+]i increase (K+-induced inhibition of EDR and [Ca2+]i increase). The Na+-K+ pump inhibitor ouabain inhibited K+-induced inhibition of EDR. Monensin (>0.1 microM) and the NCX (forward and reverse mode) inhibitors 2'4'-dichlorobenzamil (>10 microM) or Ni2+ (>100 microM) inhibited K+-induced inhibition of EDR and [Ca2+]i increase. KB-R7943 did not inhibit K+-induced inhibition at up to 10 microM but did at 30 microM. In current-clamped MAECs, an increase in [K+]o from 6 to 12 mM depolarized the membrane potential, which was inhibited by ouabain, Ni2+, or KB-R7943. In aortic rings, the concentration of cGMP was significantly increased by acetylcholine and decreased on increasing [K+]o from 6 to 12 mM. This decrease in cGMP was significantly inhibited by pretreating with ouabain (100 microM), Ni2+ (300 microM), or KB-R7943 (30 microM). These results suggest that activation of the forward mode of NCX after Na+-K+ pump activation inhibits Ca2+ mobilization in endothelial cells, thereby modulating vasomotor tone.     

Na+/K+ ATPase and its functional coupling with Na+/Ca2+ exchanger in mouse embryonic stem cells during differentiation into cardiomyocytes

Cardiomyocytes derived from mouse embryonic stem (mES) cells have been demonstrated to exhibit a time-dependent expression of ion channels and signal transduction pathways in electrophysiological studies. However, ion transporters, such as Na+/K+ ATPase (Na+ pump) or Na+/Ca2+ exchanger, which play crucial roles for cardiac function, have not been well studied in this system. In this study, we investigated the functional expression of Na+/K+ ATPase and Na+/Ca2+ exchanger in mES cells during in vitro differentiation into cardiomyocytes, as well as the functional coupling between the two transporters. By measuring [Na+]i and Na+ pump current (Ip), it was shown that an ouabain-high sensitive Na+/K+ ATPase was expressed functionally in undifferentiated mES cells and these activities increased during a time course of differentiation. Using RT-PCR, the expression of mRNA for alpha1-subunit and alpha3-subunit of the Na+/K+ ATPase could be detected in both undifferentiated mES cells and derived cardiomyocytes. In contrast alpha2-subunit mRNA could be detected only in derived cardiomyocytes but not in undifferentiated mES cells. mRNA for the Na+/Ca2+ exchanger 1 isoform (NCX1) could be detected in undifferentiated mES cells and its expression levels seemed to gradually increase throughout the differentiation accompanied by increasing its Ca2+ extrusion function. At the middle stages of differentiation (after 10-day induction), more than 75% derived cardiomyocytes exhibited [Ca2+]i oscillations by blocking of Na+/K+ ATPase, suggesting the functional coupling with Na+/Ca2+ exchanger. From these results and RT-PCR analysis, we conclude that alpha2-subunit Na+/K+ ATPase mainly contributes to establish the functional coupling with NCX1 at the middle stages of differentiation of cardiomyocytes.     

Sodium ion influx in proliferating lymphocytes: an early component of the mitogenic signal

Stimulation of pig peripheral blood lymphocytes with concanavalin A (Con A) provoked a rapid increase (two- to threefold) in the rate of ouabain-inhibitable K+ uptake observable within 3-10 min of stimulation with mitogen. At least two phases can be distinguished in the activation of the Na+/K+ pump: the early phase (till 3 h) is characterized by an unaltered number of ouabain binding sites and the later phase (noted at 5 h) by an increased number of such sites. Both K+ efflux and influx increased to the same extent, thereby maintaining [K+]i at the same level as in resting cells (120 mM). Within 3 min of addition of mitogen, the rates of total and amiloride-inhibitable Na+ uptake went up two- and fourfold, respectively, thus resulting in rapid increase in [Na+]i from 20 to about 50 mM. Activation of the Na+/K+ pump was not observed when the cells were stimulated with Con A in low Na+ medium (9 mM), nor did the usual rise in [Na+]i occur. When monensin (30 microM), a Na+/H+ ionophore, was added to resting cells, an increase in both [Na+]i and active K+ uptake occurred in normal medium but not when cells were suspended in low Na+ isotonic buffer. Amiloride (500 microM), on the other hand, prevented both the Con A-induced increase in [Na+]i and the activation of the Na+/K+ pump. Despite complete inhibition of the Na+,K+-ATPase in the presence of ouabain (1 mM), Con A activated the amiloride-inhibitable Na+ uptake in the usual way. In mouse splenocytes stimulated with Con A, there was also a parallel rise in both [Na+]i and active K+ uptake but this took considerably longer to occur than was the case in pig peripheral blood lymphocytes. Increase in both ionic fluxes, the former passive and the latter active, is essential to the entry and maintenance of the cells in proliferative cycle.     

Inversion of the intracellular Na+/K+ ratio blocks apoptosis in vascular smooth muscle at a site upstream of caspase-3.

Long term elevation of the intracellular Na+/K+ ratio inhibits macromolecule synthesis and proliferation in the majority of cell types studied so far, including vascular smooth muscle cells (VSMC). We report here that inhibition of the Na+,K+ pump in VSMC by ouabain or a 1-h preincubation in K+-depleted medium attenuated apoptosis triggered by serum withdrawal, staurosporine, or okadaic acid. In the absence of ouabain, both DNA degradation and Caspase-3 activation in VSMC undergoing apoptosis were insensitive to modification of the extracellular Na+/K+ ratio as well as to hyperosmotic cell shrinkage. In contrast, protection of VSMC from apoptosis by ouabain was abolished under equimolar substitution of Na+o with K+o, showing that the antiapoptotic action of Na+,K+ pump inhibition was caused by inversion of the intracellular Na+/K+ ratio. Unlike VSMC, the same level of increment of the [Na+]i/[K+]i ratio caused by a 2-h preincubation of Jurkat cells with ouabain did not affect chromatin cleavage and Caspase-3 activity triggered by treatment with Fas ligand, staurosporine, or hyperosmotic shrinkage. Thus, our results show for the first time that similar to cell proliferation, maintenance of a physiologically low intracellular Na+/K+ ratio is required for progression of VSMC apoptosis.     

Inhibition of endothelium-dependent vasorelaxation by extracellular K(+): a novel controlling signal for vascular contractility

The effects of extracellular K+ on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) were examined in mouse aorta, mouse aorta endothelial cells (MAEC), and human umbilical vein endothelial cells (HUVEC). In mouse aortic rings precontracted with prostaglandin F2alpha or norepinephrine, an increase in extracellular K+ concentration ([K+]o) from 6 to 12 mM inhibited EDR concentration dependently. In endothelial cells, an increase in [K+]o inhibited the agonist-induced [Ca2+]i increase concentration dependently. Similar to K+, Cs+ also inhibited EDR and the increase in [Ca2+]i concentration dependently. In current-clamped HUVEC, increasing [K+]o from 6 to 12 mM depolarized membrane potential from -32.8 +/- 2.7 to -8.6 +/- 4.9 mV (n = 8). In voltage-clamped HUVEC, depolarizing the holding potential from -50 to -25 mV decreased [Ca2+]i significantly from 0.95 +/- 0.03 to 0.88 +/- 0.03 microM (n = 11, P < 0.01) and further decreased [Ca2+]i to 0.47 +/- 0.04 microM by depolarizing the holding potential from -25 to 0 mV (n = 11, P < 0.001). Tetraethylammonium (1 mM) inhibited EDR and the ATP-induced [Ca2+]i increase in voltage-clamped MAEC. The intermediate-conductance Ca2+-activated K+ channel openers 1-ethyl-2-benzimidazolinone, chlorozoxazone, and zoxazolamine reversed the K+-induced inhibition of EDR and increase in [Ca2+]i. The K+-induced inhibition of EDR and increase in [Ca2+]i was abolished by the Na+-K+ pump inhibitor ouabain (10 microM). These results indicate that an increase of [K+]o in the physiological range (6-12 mM) inhibits [Ca2+]i increase in endothelial cells and diminishes EDR by depolarizing the membrane potential, decreasing K+ efflux, and activating the Na+-K+ pump, thereby modulating the release of endothelium-derived vasoactive factors from endothelial cells and vasomotor tone.     

Nuclear magnetic resonance studies of sodium/calcium exchange in frog perfused, beating hearts

This study explores the effect of extracellular Ca2+ concentration ([Ca2+]o), on the intracellular Na+ concentration ([Na+]i), in frog intact hearts using nuclear magnetic resonance spectroscopy, which allows for the measurement of [Na+]i in perfused, beating hearts. Decreases in [Ca2+]o yielded marked increases in [Na+]i. A similar effect was seen during inhibition of the Na+/K+ pump and was fully reversible. This sensitivity of [Na+]i to [Ca2+]o, previously observed using microelectrodes, supports a crucial physiological role for Na+/Ca2+ exchange in frog intact, beating hearts.     

Effect of inhibition of Na+/K(+)-ATPase on the vasopressin-induced increase in intracellular Na+ concentration in vascular smooth muscle cells.

The effect of inhibition of Na+/K(+)-ATPase by ouabain on the arginine vasopressin (AVP)-induced increase in intracellular Na+ concentration [( Na+]i) was examined in cultured rat vascular smooth muscle cells (VSMC) by the direct measurement of [Na+]i using a fluorescent indicator dye. AVP at a concentration of 1 x 10(-9) M or higher increased [Na+]i in a dose-dependent manner in cultured rat VSMC. The preincubation of cells with 1 x 10(-4) M ouabain for 1 hr at 37 degrees C did not affect the basal [Na+]i but enhanced the 1 x 10(-6) M AVP-induced increase in [Na+]i. The preincubation was not necessary because similar results were obtained after the simultaneous administration of AVP and ouabain. The treatment with ouabain did not affect the intracellular pH changes induced by AVP. These results therefore indicate that the inhibition of Na+/K(+)-ATPase enhances the AVP-induced increase in [Na+]i by decreasing cellular Na+ efflux in cultured rat VSMC.     

Fluorescence lifetime microscopy of the Na+ indicator Sodium Green in HeLa cells

This study investigates the usefulness of lifetime measurements of Sodium Green for evaluating intracellular Na+ concentration ([Na+]i) in HeLa cells. Frequency-domain lifetime measurements are performed in HeLa cells and in different buffer solutions (with and without K+ and bovine serum albumin). In all cases, the fluorescence decays of Sodium Green are multiexponential, with decay times independent of [Na+]. Three relaxation times are found in the various buffer solutions. Binding of the indicator to albumin results in an increase in the long and intermediate decay times. For Sodium Green inside HeLa cells, the intensity decay can be approximated by a biexponential. The ratio of the fractional intensity of the long decay time (tau2 = 2.4 +/- 0.2 ns) to that of the short component (tau1 = 0.4 +/- 0.1 ns) increases with [Na+]i. The changes in fluorescence decay with [Na+] are significantly less pronounced in cells as compared with the buffer solutions. Similar values for the resting [Na+]i were estimated from lifetime measurements of Sodium Green and from ratiometric measurements using SBFI. Alternatively, [Na+]i can be monitored by measuring only the phase angle at the modulation frequency of 160 MHz. The usefulness of this latter approach is demonstrated by following the changes in [Na+]i induced by reversible inhibition of the Na+/K+ pump.     

Relationships between the neuronal sodium/potassium pump and energy metabolism. Effects of K+, Na+, and adenosine triphosphate in isolated brain synaptosomes

The relationships between Na/K pump activity and adenosine triphosphate (ATP) production were determined in isolated rat brain synaptosomes. The activity of the enzyme was modulated by altering [K+]e, [Na+]i, and [ATP]i while synaptosomal oxygen uptake and lactate production were measured simultaneously. KCl increased respiration and glycolysis with an apparent Km of about 1 mM which suggests that, at the [K+]e normally present in brain, 3.3-4 mM, the pump is near saturation with this cation. Depolarization with 6-40 mM KCl had negligible effect on ouabain-sensitive O2 uptake indicating that at the voltages involved the activity of the Na/K ATPase is largely independent of membrane potential. Increases in [Na+]i by addition of veratridine markedly enhanced glycoside-inhibitable respiration and lactate production. Calculations of the rates of ATP synthesis necessary to support the operation of the pump showed that greater than 90% of the energy was derived from oxidative phosphorylation. Consistent with this: (a) the ouabain-sensitive Rb/O2 ratio was close to 12 (i.e., Rb/ATP ratio of 2); (b) inhibition of mitochondrial ATP synthesis by Amytal resulted in a decrease in the glycoside-dependent rate of 86Rb uptake. Analyses of the mechanisms responsible for activation of the energy-producing pathways during enhanced Na and K movements indicate that glycolysis is predominantly stimulated by increase in activity of phosphofructokinase mediated via a rise in the concentrations of adenosine monophosphate [AMP] and inorganic phosphate [Pi] and a fall in the concentration of phosphocreatine [PCr]; the main moving force for the elevation in mitochondrial ATP generation is the decline in [ATP]/[ADP] [Pi] (or equivalent) and consequent readjustments in the ratio of the intramitochondrial pyridine nucleotides [( NAD]m/[NADH]m). Direct stimulation of pyruvate dehydrogenase by calcium appears to be of secondary importance. It is concluded that synaptosomal Na/K pump is fueled primarily by oxidative phosphorylation and that a fall in [ATP]/[ADP][Pi] is the chief factor responsible for increased energy production.     

Agonist-specific regulation of [Na+]i in pancreatic acinar cells

In a companion paper (Zhao, H., and S. Muallem. 1995), we describe the relationship between the major Na+,K+, and Cl- transporters in resting pancreatic acinar cells. The present study evaluated the role of the different transporters in regulating [Na+]i and electrolyte secretion during agonist stimulation. Cell stimulation increased [Na+]i and 86Rb influx in an agonist-specific manner. Ca(2+)-mobilizing agonists, such as carbachol and cholecystokinin, activated Na+ influx by a tetraethylammonium-sensitive channel and the Na+/H+ exchanger to rapidly increase [Na+]i from approximately 11.7 mM to between 34 and 39 mM. As a consequence, the NaK2Cl cotransporter was largely inhibited and the activity of the Na+ pump increased to mediate most of the 86Rb(K+) uptake into the cells. Secretin, which increases cAMP, activated the NaK2Cl cotransporter and the Na+/H+ exchanger to slowly increase [Na+]i from approximately 11.7 mM to an average of 24.6 mM. Accordingly, secretin increased total 86Rb uptake more than the Ca(2+)- mobilizing agonists and the apparent coupling between the NaK2Cl cotransport and the Na+ pump. All the effects of secretin could be attributed to an increase in cAMP, since forskolin affected [Na+]i and 86Rb fluxes similar to secretin. The signaling pathways mediating the effects of the Ca(2+)-mobilizing agonists were less clear. Although an increase in [Ca2+]i was required, it was not sufficient to account for the effect of the agonists. Activation of protein kinase C stimulated the NaK2Cl cotransporter to increase [Na+]i and 86Rb fluxes without preventing the inhibition of the cotransporter by Ca(2+)-mobilizing agonists. The effects of the agonists were not mediated by changes in cell volume, since cell swelling and shrinkage did not reproduce the effect of the agonists on [Na+]i and 86Rb fluxes. The overall findings of the relationships between the various Na+,K+, and Cl- transporters in resting and stimulated pancreatic acinar cells are discussed in terms of possible models of fluid and electrolyte secretion by these cells.     

Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells

Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]i from 114.3+/-15.4 nM to 531.1+/-71.9 nM (n=6) and also increased frequency of K+ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca2+]i, the transient inhibition of K+ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5+/-122.7 nM). We concluded that H2O2 not only activates K+ATP channels in association with metabolic inhibition, but also increases partly the Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.     

Na/K pump and intracellular monovalent cations: novel mechanism of excitation-transcription coupling involved in inhibition of apoptosis

The Na/K pump plays a key role in the regulation of the intracellular concentrations of monovalent cations and related cell function leading to electrogenesis and excitation-contraction coupling. We focus this review on the analysis of recent data showing that (i) inhibition of the Na/K pump triggers a signaling cascade independently of modulation of the intracellular [Na+]i/[K+]i ratio; (ii) elevation of [Na+]i under sustained inhibition of the Na/K pump leads to expression of a set of genes by [Ca2+]i-dependent and independent pathways; (iii) [Na+]i-sensitive genes are involved in the inhibition of programmed cell death (apoptosis) in vascular smooth muscle cells.     

Reaccumulation of [K+]o in the toad retina during maintained illumination

Using K+-selective microelectrodes, [K+]o was measured in the subretinal space of the isolated retina of the toad, Bufo marinus. During maintained illumination, [K+]o fell to a minimum and then recovered to a steady level that was approximately 0.1 mM below its dark level. Spatial buffering of [K+]o by Müller (glial) cells could contribute to this reaccumulation of K+. However, superfusion with substances that might be expected to block glial transport of K+ had no significant effect upon the reaccumulation of K+. These substances included blockers of gK (TEA+, Cs+, Rb+, 4-AP) and a gliotoxin (alpha AAA). Progressive slowing of the rods' Na+/K+ pump (perhaps caused by a light-evoked decrease in [Na+]i) also could contribute to this reaccumulation of K+ by reducing the uptake of K+ from the subretinal space. As evidence for a major contribution by this mechanism, treatments designed to prevent such slowing of the pump reversibly blocked reaccumulation. These treatments included superfusion with 2 microM ouabain, or lowering [K+]o, PO2, or temperature. It is likely that such treatments inhibit the pump, increase [Na+]i, and attenuate any light-evoked decrease in [Na+]i. The results are consistent with the following hypothesis. At light onset, the decrease in rod gNa will reduce the Na+ influx and the resulting rod hyperpolarization will reduce the K+ efflux. In combination with these reduced passive fluxes, the continuing active fluxes will lower both [K+]o and [Na+]i, which in turn will inhibit the pump. In support of this hypothesis, the solutions to a pair of coupled differential equations that model changes in both [K+]o and [Na+]i match quantitatively the time course of the observed changes in [K+]o during and after maintained illumination for all stimuli examined.     

Saturation of the internal sodium site of the sodium pump can distort estimates of potassium affinity.

The Na+/K+ exchange pump in cardiac Purkinje strands has been well studied with the voltage clamp and Na+-selective microelectrodes. Models describing the observed results suggest that the pump rate can be considered proportional to [Na+]i over the range examined and depends on external [K+] in accordance with Michaelis-Menten kinetics. Estimates of the external [K+] that achieves a half-maximal pump rate (Km) range from 0.9 to 6.3 mM depending on the preparation and method of estimation. Here we show that much of the variability in the estimates of the Km can be eliminated when saturation of the internal Na+ pump site is taken into account. If the half-activation concentration for saturation of this Na+ site is sufficiently high (greater than 20 mM), removal of intracellular Na+ in response to a Na+ load will approximate first-order kinetics. Under these conditions however, Na+ saturation will nevertheless cause large systematic errors in estimates of the K+ dependence of pump activity.     

Induction of system A amino acid transport through long-term treatment with ouabain: correlation with increased (Na+/K+)-ATPase activity

Mouse embryo fibroblast cells (C3H-10T1/2) and the methylcholanthrene-transformed derivative (MCA-10T1/2) were treated with basal modified Eagle's medium (BME) containing 10% fetal bovine serum and varying concentrations of ouabain ranging from 0.05 mM to 0.7 mM for 16 h in culture. After replacing the ouabain-containing medium with Earl's balanced salts solution, System A amino acid transport activity increased from approximately 40 to 500 pmol AIB accumulated.mg protein-1.min-1 in the C3H-10T1/2 cells and from approximately 300 to 700 pmol AIB accumulated.mg protein-1.min-1 in the MCA-10T1/2 cells. The (Na+/K+)-ATPase pump activity also increased from approximately 12 to 46 nmol Rb+ accumulated.mg protein-1.min-1 in the normal cells and from approximately 20 to 42 nmol Rb+ accumulated.mg protein-1.min-1 in the transformed cells. System A and the (Na+/K+)ATPase activity were maximally increased at approximately 0.4-0.6 mM ouabain in the normal cells in contrast to the transformed cells which were maximally stimulated at a concentration of approximately 0.2 mM ouabain. This treatment with ouabain increased the [Na+]i/[K+]i as measured by atomic absorption spectroscopy, and thereby decreased the Na+ and K+ electrochemical gradients. Our data show that the internal ion gradients inverted at a lower concentration of ouabain in the transformed cells compared to the normal cells. The ouabain-induced increase in pump and System A activity shown here was used as a tool to further investigate the coordinated ion transport regulation in the control of cell growth.     

Transport by the (Na+,K+) ATPase: modulation by differentiation inducers and inhibition of protein synthesis in the MDCK kidney epithelial cell line

MDCK kidney epithelial cell cultures exposed to the differentiation inducer hexamethylene bisacetamide (HMBA) for 24 hours exhibited a 50% decrease in transport activity per (Na+,K+)-ATPase molecule (turnover number) but an unchanged number of pump sites (Kennedy and Lever, 1984). Inhibition of protein synthesis by either 10 microM cycloheximide or 2 microM emetine blocked the inhibitory effects of HMBA on Na+/K+ pump efficiency assessed by measurements of [3H]-ouabain binding to intact cells, (Na+,K+) ATPase activity of detergent-activated cell extracts, and ouabain-sensitive Rb+ uptake. In the absence of inducer treatment, inhibition of protein synthesis increased Na+/K+ pump turnover number by twofold while maintaining Na+/K+ pump activity per cell at a constant level. Intracellular Na+ levels were decreased after cycloheximide treatment; therefore, pump stimulation was not due to substrate effects. Furthermore, cycloheximide effects of Rb+ uptake could be dissociated from effects on tight junctions. These observations suggest that the transport activity of the (Na+,K+) ATPase is tightly regulated by factors dependent on protein synthesis.     

Activation of AMPA/kainate receptors but not acetylcholine receptors causes Mg2+ influx into Retzius neurones of the leech Hirudo medicinalis

In Retzius neurones of the medicinal leech, Hirudo medicinalis, kainate activates ionotropic glutamate receptors classified as AMPA/kainate receptors. Activation of the AMPA/kainate receptor-coupled cation channels evokes a marked depolarization, intracellular acidification, and increases in the intracellular concentrations of Na+ ([Na+]i) and Ca2+. Qualitatively similar changes are observed upon the application of carbachol, an activator of acetylcholine receptor-coupled cation channels. Using multibarrelled ion-selective microelectrodes it was demonstrated that kainate, but not carbachol, caused additional increases in the intracellular free Mg2+ concentration ([Mg2+]i). Experiments were designed to investigate whether this kainate-induced [Mg2+]i increase was due to a direct Mg2+ influx through the AMPA/kainate receptor-coupled cation channels or a secondary effect due to the depolarization or the ionic changes. It was found that: (a) Similar [Mg2+]i increases were evoked by the application of glutamate or aspartate. (b) All kainate-induced effects were inhibited by the glutamatergic antagonist DNQX. (c) The magnitude of the [Mg2+]i increases depended on the extracellular Mg2+ concentration. (d) A reduction of the extracellular Ca2+ concentration increased kainate-induced [Mg2+]i increases, excluding possible Ca2+ interference at the Mg2+-selective microelectrode or at intracellular buffer sites. (e) Neither depolarizations evoked by the application of 30 mM K+, nor [Na+]i increases induced by the inhibition of the Na+/K+ ATPase caused comparable [Mg2+]i increases. (f) Inhibitors of voltage-dependent Ca2+ channels did not affect the kainate-induced [Mg2+]i increases. Moreover, previous experiments had already shown that intracellular acidification evoked by the application of 20 mM propionate did not cause changes in [Mg2+]i. The results indicate that kainate-induced [Mg2+]i increases in leech Retzius neurones are due to an influx of extracellular Mg2+ through the AMPA/kainate receptor-coupled cation channel. Mg2+ may thus act as an intracellular signal to distinguish between glutamatergic and cholinergic activation of leech Retzius neurones.     

Platelet free calcium concentrations measured with fura-2 are influenced by the transmembrane sodium gradient

The influence of the transmembrane Na+ gradient on the intracellular free calcium concentration, [Ca2+]i, was studied in Sepharose gel-filtered platelets from healthy human subjects, using the Ca-sensitive fluorescent dye, fura-2. Raising the internal Na+ concentration, [Na+]i, by Na+ pump inhibition with 0.05 mM ouabain, without changing external Na+ did not cause a significant increase in [Ca2+]i. Substitution of extracellular Na+ by iso-osmolar sucrose induced a rapid (half-time about 2 min) and significant rise in [Ca2+]i; this effect was amplified in Na-loaded platelets. Partial restitution of external Na+ in these cells with increased [Ca2+]i promoted a significant and rapid Na+ concentration-dependent fall in [Ca2+]i; little decline in [Ca2+]i was observed if K+ was used instead of Na+. These observations represent in vitro evidence for the existence of a Na/Ca exchange mechanism in human platelets that may, in vivo, participate in the control of [Ca2+]i.     

Inhibition by lead ion of Electrophorus electroplax (Na+ + K+)-adenosine triphosphatase and K+-p-nitrophenylphosphatase.

Inorganic lead ion in micromolar concentrations inhibits Electrophorus electroplax microsomal (Na+ + K+)-adenosine triphosphatase ((Na+ + K+)-ATPase) and K+-p-nitrophenylphosphatase (NPPase). Under the same conditions, the same concentrations of PbCl2 that inhibit ATPase activity also stimulate the phosphorylation of electroplax microsomes in the absence of added Na+. Enzyme activity is protected from inhibition by increasing concentrations of microsomes, ATP, and other metal ion chelators. The kinetics follow the pattern of a reversible noncompetitive inhibitor. No kinetic evidence is elicited for interactions of Pb2+ with Na+, K+, Mg2+, ATP, or p-nitrophenylphosphate. Na+- ATPase, in the absence of K+, and (Na+ + K+)-NPPase activity at low [K+] are also inhibited. ATP inhibition of NPPase is not reversed by Pb2+. The calculated concentrations of free [Pb2+] that produce 50% inhibition are similar for ATPase and NPPase activities. Pb2+ may act at a single independent binding site to produce both stimulation of the kinase and inhibition of the phosphatase activities.