Agonist-dependent regulation of renal Na+,K+-ATPase activity is modulated by intracellular sodium concentration.

We tested the hypothesis that the level of intracellular sodium modulates the hormonal regulation of the Na(+),K(+)-ATPase activity in proximal tubule cells. By using digital imaging fluorescence microscopy of a sodium-sensitive dye, we determined that the sodium ionophore monensin induced a dose-specific increase of intracellular sodium. A correspondence between the elevation of intracellular sodium and the level of dopamine-induced inhibition of Na(+),K(+)-ATPase activity was determined. At basal intracellular sodium concentration, stimulation of cellular protein kinase C by phorbol 12-myristate 13-acetate (PMA) promoted a significant increase in Na(+),K(+)-ATPase activity; however, this activation was gradually reduced as the concentration of intracellular sodium was increased to become a significant inhibition at concentrations of intracellular sodium higher than 16 mm. Under these conditions, PMA and dopamine share the same signaling pathway to inhibit the Na(+),K(+)-ATPase. The effects of PMA and dopamine on the Na(+),K(+)-ATPase activity and the modulation of these effects by different intracellular sodium concentrations were not modified when extracellular and intracellular calcium were almost eliminated. These results suggest that the level of intracellular sodium modulates whether hormones stimulate, inhibit, or have no effect on the Na(+),K(+)-ATPase activity leading to a tight control of sodium reabsorption.     

竹红菌乙素对人红细胞Na~+-K~+ATPase和钠通透性光敏损伤的研究

本文用NMR和生化方法研究了竹红菌乙素对人红细胞膜Na~+-K~+ATPase和钠通透性的光敏损伤。结果表明:在通常情况下,可同时观察到乙素对Na~+-K~+ATPase和钠通透性的光敏损伤。比较乙素、甲素、原卟啉和胆红素对上述两项指标的光敏能力,发现乙素对Na~+-K~+ATPase损伤能力与甲素和原卟啉相当,比胆红素大,对钠通透性的损伤大于其它几种敏化剂。实验指出,Na~+-K~+ATPase活力下降比钠通透性增加敏感。在乙素光敏作用时,加入Vit E可基术上保持胞内钠离子浓度不变,但无法使Na~+-K~+ATPase活力不损伤,这表明膜磷脂的结构完整对保持胞内钠浓度比较重要。对照试验指出乙素可使Na~+-K~+ATPase暗失活,这可能是经乙素介导的,由膜还原物质向氧的电子传递生成活性氧自由基攻击靶分子所致。     

The Na+/H+ exchanger isoform 1

The Na+/H+ exchanger (NHE) isoform 1 is a ubiquitously expressed integral membrane protein which regulates intracellular pH in mammalian cells. Nine isoforms of the Na+/H+ exchanger have been identified. The isoform first discovered has two domains: an N-terminal membrane domain containing approximately 500 amino acids and a C-terminal regulatory domain containing approximately 315 amino acids. The exchanger, which resides in the plasma membrane, exchanges an intracellular proton for an extracellular sodium, thereby regulating intracellular pH. It is involved in cell growth and differentiation, cell migration, and regulation of sodium fluxes. The Na+/H+ exchanger plays an important role in myocardial damage during ischemia and reperfusion and has recently been implicated as a mediator of cardiac hypertrophy. Inhibitors of the Na+/H+ exchanger, which may prove useful in the clinical treatment of these conditions, are currently being developed and clinical trials are underway.     

Ionic, electrical and water balance in the animal cell. The system with active cation transport, Goldman's channels and the Na + K +2Cl-type symport]

The relations between intracellular potassium, sodium water content and resting potential on the one hand and the ion transport parameters and intracellular electrical charge on the other hand were computed for a model of animal cell with a several ion transporters and variable intracellular charge. The case of the balanced ion distribution is considered. The results are presented in a graphical form.     

Modification of the internal pH sensitivity of the Na+/H+ antiporter by parathyroid hormone in a cultured renal cell line

Sodium-proton antiporter activity can be modulated through changes Vmax and/or intracellular proton sensitivity of the antiporter. To characterize a parathyroid hormone (PTH)-induced decrease in antiporter activity in a continuous renal cell line (opossum kidney cells), the extracellular sodium and intracellular proton dependence of amiloride-inhibitable 22Na uptake was studied. The Km for extracellular sodium at intracellular pH 6.32 was 28 mM and was unaltered by PTH, whereas the Vmax was decreased by 26%. When intracellular pH was set over the range 5.87-7.57 by the potassium-nigericin method, antiporter activity increased as intracellular pH decreased. Hill analysis revealed Hill coefficients of 1.25 and 1.01 and half-maximal antiporter activity at intracellular pH values of 6.90 and 6.35 for control and PTH-treated cells, respectively. PTH decreased the apparent Vmax at low pH by 15% and the intracellular pH at which Na+/H+ exchange is half-maximal by 0.55 pH units.     

Cytochalasin B induced changes in cytoplasmic Na+/K+ balance in 2-cell mouse embryo

This work was performed to study changes in intracellular elemental (Na/K) concentrations caused by Cytochalasin B in two-cell mouse embryo using Electron Probe Microanalysis. The presence of Cytochalasin B is required to transfer a somatic cell nuclear into an early embryo cell. The direct effect of this chemical is cytoskeleton transformation, which would be able to cause the increase of potassium channel activity resulting in cytoplasmic Na/K imbalance. In our study Cytochalasin B was shown to decrease the intracellular sodium concentration. The Na/K balance in the cytoplasm of mouse embryos reverted to its intact level after treatment them with Cytochalasin B free Dulbecco's solution. Possible mechanisms responsible for the changes in the intracellular sodium concentration observed in the embryo cells are discussed.     

竹红菌素对人红细胞Na^＋—K^＋ATPase和钠通透性光敏损伤的研究

The hypocrellin B (HB)-sensitized photodamage on Na(+)-K+ ATPase and sodium permeability of human erythrocytes by means of NMR and biochemical techniques was studied in this paper. The decrease of the enzyme activity and increase of intracellular sodium concentration were usually observed simultaneously. The evidences suggested that the integrality of membrane phospholipid played an important role in maintaining the physiological sodium content of erythrocytes. The loss of the enzyme activity was a sensitive index compared with the increase of intracellular Na+ concentration during the photosensitization. From the comparison tests among HB, HA, protoporphyrin and bilirubin, we found that HB had more ability to increasing intracellular Na+ concentration than the other photosensitization even though the photodamage on the enzyme activity caused by HB, HA, and protoporphyrin were nearly the same. Besides the photoinactivation of Na(+)-K+ ATPase induced by HB and light, the enzyme was also inactivated in the medium containing HB in absence of light. The active oxygen radicals generated though HB mediated redox-cycling might be involved in the dark inactivation of the enzyme.     

Short-Term Regulation of the Proximal Tubule Na+,K+-ATPase: Increased/Decreased Na+,K+-ATPase Activity Mediated by Protein Kinase C Isoforms

In different species and tissues, a great variety of hormones modulate Na⁺,K⁺-ATPase activity in a short-term fashion. Such regulation involves the activation of distinct intracellular signaling networks that are often hormone- and tissue-specific. This minireview focuses on our own experimental observations obtained by studying the regulation of the rodent proximal tubule Na⁺,K⁺-ATPase. We discuss evidence that hormones responsible for regulating kidney proximal tubule sodium reabsorption may not affect the intrinsic catalytic activity of the Na⁺,K⁺-ATPase, but rather the number of active units within the plasma membrane due to shuttling Na⁺,K⁺-ATPase molecules between intracellular compartments and the plasma membrane. These processes are mediated by different isoforms of protein kinase C and depend largely on variations in intracellular sodium concentrations.     

Biology of the 2Na+/1H+ antiporter in invertebrates

The functional expression of membrane transport proteins that are responsible for exchanging sodium and protons is a ubiquitous phenomenon. Among vertebrates the Na+/H+ antiporter occurs in plasma membranes of polarized epithelial cells and non-polarized cells such as red blood cells, muscle cells, and neurons, and in each cell type the transporter exchanges one sodium for one hydrogen ion, is inhibited by amiloride, and regulates intracellular pH and sodium concentration within tight limitations. In polarized epithelial cells this transporter occurs in two isoforms, each of which is restricted to either the brush border or basolateral cell membrane, and perform somewhat different tasks in the two locations. In prokaryotic cells, sodium/proton exchange occurs by an electrogenic 1Na+/2H+ antiporter that is coupled to a primary active proton pump and together these two proteins are capable of tightly regulating the intracellular concentrations of these cations in cells that may occur in environments of 4 M NaCl or pH 10-12. Invertebrate epithelial cells from the gills, gut, and kidney also exhibit electrogenic sodium/proton exchange, but in this instance the transport stoichiometry is 2Na+/1H+. As with vertebrate electroneutral Na+/H+ exchange, the invertebrate transporter is inhibited by amiloride, but because of the occurrence of two external monovalent cation binding sites, divalent cations are able to replace external sodium and also be transported by this system. As a result, both calcium and divalent heavy metals, such as zinc and cadmium, are transported across epithelial brush border membranes in these animals and subsequently undergo a variety of biological activities once accumulated within these cells. Absorbed epithelial calcium in the crustacean hepatopancreas may participate in organismic calcium balance during the molt cycle and accumulated heavy metals may undergo complexation reactions with intracellular anions as a detoxification mechanism. Therefore, while the basic process of sodium/proton exchange may occur in invertebrate cells, the presence of the electrogenic 2Na+/1H+ antiporter in these cells allows them to perform a wide array of functions without the need to develop and express additional specialized transport proteins. J. Exp. Zool. 289:232-244, 2001.     

The effects of a low extracellular concentration of potassium on the activity and numbers of Na+/K+ pumps in an EB-virus transformed human lymphocyte cell line

The BM1A EB-virus transformed human lymphocyte cell line contains approximately 950,000 Na+/K(+)-ATPase sites per cell. The turnover number of each site is approx. 2240 molecules of rubidium per min. When cells are exposed to a low extracellular concentration of potassium the intracellular concentration of sodium rises, and the cells respond in the short term by increasing the Vmax of 86Rb+ uptake. In the longer term the cells respond by increasing both the Vmax of 86Rb+ uptake and the Bmax of [3H]ouabain binding. The suggestion that increases in the intracellular concentration of sodium is responsible for these changes is supported by the finding that monensin, which increases intracellular sodium without affecting intracellular potassium, is capable of inducing both the short- and long-term changes associated with a low external concentration of potassium.     

Diverse residues of intracellular loop 5 of the Na+/H+ exchanger modulate proton sensing,expression, activity and targeting

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH (pH_i) by removing a single intracellular proton in exchange for one extracellular sodium ion. It is involved in cardiac hypertrophy and ischemia reperfusion damage to the heart and elevation of its activity is a trigger for breast cancer metastasis. NHE1 has an extensive 500 amino acid N-terminal membrane domain that mediates transport and consists of 12 transmembrane segments connected by intracellular and extracellular loops. Intracellular loops are hypothesized to modulate the sensitivity to pH_i. In this study, we characterized the structure and function of intracellular loop 5 (IL5), specifically amino acids 431–443. Mutation of eleven residues to alanine caused partial or nearly complete inhibition of transport; notably, mutation of residues L432, T433, I436, N437, R440 and K443 demonstrated these residues had critical roles in NHE1 function independent of effects on targeting or expression. The nuclear magnetic resonance (NMR) solution spectra of the IL5 peptide in a membrane mimetic sodium dodecyl sulfate solution revealed that IL5 has a stable three-dimensional structure with substantial alpha helical character. NMR chemical shifts indicated that K438 was in close proximity with W434. Overall, our results show that IL5 is a critical, intracellular loop with a propensity to form an alpha helix, and many residues of this intracellular loop are critical to proton sensing and ion transport.     

Voltage dependence of H+ buffering mediated by sodium bicarbonate cotransport expressed in Xenopus oocytes

The electrogenic sodium bicarbonate cotransporter (NBCe1) is expressed in many epithelial cells and, in the brain, in glial cells. Little is known about the physiological significance of the NBCe1 for proton homeostasis and for other acid/base-coupled transporters in these cells. We have measured the voltage-dependent transport activity of an NBC from human kidney, type hkNBCe1, expressed in oocytes of the frog Xenopus laevis, by recording membrane current and the changes in intracellular pH and sodium at different membrane potentials between -20 and -100 mV. The apparent intracellular buffer capacity was increased and became dependent upon membrane voltage when the NBCe1 was expressed; the measured buffer capacity increased by up to 7 mm/10 mV of membrane depolarization. Lactate transport by the electroneutral monocarboxylate transporter became enhanced and dependent upon membrane potential, when the monocarboxylate transporter (isoform 1) was co-expressed with NBCe1 in oocytes. Our results indicate that the electrogenic NBCe1 renders the cell membrane potential an effective regulator of intracellular H(+) buffering and acid/base-coupled metabolite transport.     

Role of Ca2+ and prostaglandin in regulation of active Na+ transport in frog skin

1. The role of prostaglandins and intracellular Ca2+ in regulation of active transepithelial sodium transport in frog skin were studied by examinations of effects of the calcium ionophore A23187 on short-circuit current (SCC) and intracellular voltage. 2. A23187 and arachidonic acid induced a marked increase in both SCC and prostaglandin E2 synthesis. 3. In indomethacin treated skins A23187 did not stimulate but on the contrary inhibited the basal SCC. 4. The A23187-induced increase in SCC was associated with a decrease in the fractional resistance of the apical membrane and a depolarization of the cells. 5. In skins pretreated with indomethacin, the A23187 induced inhibition of SCC coincided with a slight hyperpolarization of the cellular potential and an increase in fractional resistance of the apical membrane.     

Characterization of Na+/H+ exchange in a rabbit corneal epithelial cell line (SIRC)

Continuous intracellular pH (pHi) measurements were performed in SIRC rabbit corneal epithelial cells using the pH-sensitive absorbance of intracellularly trapped 5(and 6)-carboxy-4',5'-dimethylfluorescein. Steady-state pHi in nominally bicarbonate free Ringer's solution averaged 6.87 +/- 0.02 (mean +/- S.E., n = 53). After intracellular acidification induced by the NH4Cl-prepulse technique, there was a sodium-dependent pHi recovery towards the normal steady-state pHi. The initial pHi recovery rate was a saturable function of extracellular sodium concentration with an apparent Km for external sodium of about 25 mM and a Vmax of about 0.28 pH units/min. Virtually no pHi recovery was observed in the absence of extracellular sodium. Sodium removal during steady state acidified the cells by 0.36 +/- 0.05 pH units (mean +/- S.E., n = 13) within 5 min. There was a dose-dependent inhibition of pHi recovery after NH4Cl prepulse by amiloride with an IC50 of about 15 microM. Amiloride in a concentration of 1 mM almost completely abolished pHi recovery. Amiloride (1 mM) applied during steady state induced an intracellular acidification of 0.2 +/- 0.03 pH units (mean +/- S.E., n = 7) within 5 min. These findings suggest that a Na+/H+ exchange is present in SIRC rabbit corneal epithelial cells. Na+/H+ exchange seems to be the major process involved in pHi recovery in SIRC cells after an intracellular acid load. Na+/H+ exchange also plays a role in the maintenance of steady-state pHi.     

Na+/H+ exchangers in the human eccrine sweat duct

Using an anti-NHE1 antibody, we demonstrate the presence of a Na+/H+ exchanger of isoform 1 (NHE1) in the human eccrine sweat duct. A strong staining was observed at the basolateral membrane of the outer cell layer (NHE1basal), at the junction between inner and outer cells layers (NHE1inter), and along the lateral membranes (NHE1later) of all cells of the duct. At the luminal membrane, no staining was demonstrated either for NHE1 or NHE3. To investigate Na+/H+ mediated proton transport, straight sweat duct portions were isolated and perfused in vitro under HCO3-free conditions. In the presence of basolateral 5-ethyl-N-isopropyl amiloride (EIPA), an acidification of 0.29 +/- 0.03 pH units was observed, whereas no effect was observed with luminal EIPA. Bath sodium removal generated a stronger acidification (0.41 +/- 0.09 pH units). Removal of luminal sodium (in the absence or presence of basolateral EIPA), or low luminal chloride, led to an alkalinization, presumably due to a decrease in intracellular sodium, strongly suggesting functional activity of NHE1inter. We therefore conclude that in the sweat duct, NHE1 plays a major role in intracellular pH regulation.     

Some Observations on the Partition of Na+ and K+ into a Lipid Phase

It is likely that sodium and potassium must traverse a lipid membrane surrounding cells and that this membrane has to do with intracellular cation selection. Electrolyte theory is inadequate to predict the partition of salts of these cations between water and a lipid phase. The data obtained here demonstrate the partition and the cation selection to be a function of the anion species (or ionized lipid), the solvent and the presence of the unionized form of the lipid. Specificity in lipid partition is not synonymous with the cation specificity in precipitation of ionic crystals.     

Redistribution of intracellular Ca2+ in mitogen-stimulated human peripheral blood lymphocytes

The fluorescent probe chlortetracycline (CTC) was used to investigate redistribution of intracellular Ca2+ in concanavalin A (Con A)-stimulated human peripheral blood lymphocytes. The addition of the mitogen to CTC-equilibrated lymphocytes induced (within 10 to 15 minutes) a Con A-concentration dependent decrease in CTC fluorescence indicating the release of membrane-bound Ca2+. The effect was independent of the level of extracellular Ca2+ and could be observed in the presence of EGTA; it was suppressed by the metabolic inhibitors FCCP, antimycin and sodium cyanide. Analysis of the excitation spectra of CTC fluorescence indicated that the observed effect is caused by redistribution of intracellular Ca2+ rather than Mg2+. Thus the lectin interaction with the lymphocyte plasma membrane results in Ca2+ release into the cytosol from the intracellular stores.     

Second-site revertants of a low-sodium-affinity mutant of the Na+/H+ exchanger reveal the participation of TM4 into a highly constrained sodium-binding site

On the basis of intracellular acidifications in the presence of 30 microM cariporide, we selected a fibroblast cell line termed CR5, expressing a mutated Na(+)/H(+) exchanger NHE-1 with a low affinity for cariporide (87 +/- 11.6 microM) and extracellular sodium (248 +/- 63.7 mM). This mutated exchanger displays a Phe162Ser substitution in its fourth transmembrane segment. Using intracellular acidifications in the presence of 3 mM external sodium on the CR5 fibroblasts, we isolated two revertants which exhibited a complete recovery for sodium affinity but were still resistant to cariporide. Sequencing the cDNAs encoding these revertants revealed the presence of two mutations situated at a distant location from Phe162 in the same fourth transmembrane segment (Ile169Ser and Ile170Thr). Interestingly, introducing these two mutations in the wild-type cDNA did not result in a significant increase in affinity for sodium. Furthermore, all the mutants characterized in this study display an unchanged affinity for lithium, another transported cation. These data suggest that the mutation resulting in the low sodium affinity and the two mutations responsible for the reversion of this phenotype affect the binding of sodium itself instead of the conformational changes triggering substrate translocation. Taken together, these results allow us to propose that optimal sodium binding by the Na(+)/H(+) exchangers requires the geometrical integrity of a highly constrained sodium coordination site.     

Microelectrode study of K+ accumulation by tight epithelia: II. Effect of inhibiting transepithelial Na+ transport on reaccumulation following depletion

The effects of restoring serosal potassium to potassium-depleted toad urinary bladders have been re-examined using double-barrelled microelectrodes. The data confirm the existence of a time-lag phenomenon, a dissociation between potassium reaccumulation and restoration of short-circuit current. Returning serosal potassium stimulates an increase in intracellular potassium activity 21-26 min before any increase can be detected in short-circuit current. The reaccumulation of potassium has been further studied using split frog skin, a far more suitable preparation for electrophysiologic study than toad bladder. Under baseline short-circuited conditions, potassium is accumulated against an electrochemical gradient of 22 +/- 4 mV. Reaccumulation of potassium by potassium-depleted tissues can be blocked by inhibiting the Na,K-exchange pump with high concentrations of ouabain. On the other hand, blocking apical sodium entry by the addition of 10(-4) M amiloride to the outer bathing medium does not interfere with reaccumulation of potassium. The data support the concept that the time-lag phenomenon of toad bladder reflects stimulation of potassium reaccumulation by the sodium pump in exchange for the extrusion of excess cell sodium collected during the period of potassium depletion. This reaccumulation of potassium can proceed before the entry of significant added amounts of sodium across the apical plasma membrane.