Effects of alkali ions on the circadian leaf movements of Oxalis regnellii

The influence of alkali ions on the circadian leaf movements of Oxalis regnellii Mig. was investigated. Ions were given to the oscillating system via the transpiration stream of cut stalks in nutrient medium. Chloride solutions of Rb⁺, Cs⁺, Na⁺ and K⁺ were tested and the results compared to previously published LiCl-results. The period of the circadian leaf movements was unaffected by a continual addition of Na⁺ or K⁺ to the nutrient medium (at least up to 40 mM). Rb⁺, in the concentration of 2.5 or 5 mM, caused a shortening of the period when applied continuously. Rb⁺ concentrations up to 60 mM were tested. Cs⁺ ions caused only lengthenings of the circadian period. Cs⁺ concentrations up to 40 mM were tested. Cs⁺ resembled Li⁺ in producing period lengthenings, but was not as effective as Li⁺ when compared on a concentration basis. Toxicity of the effective ions was in the following order: Li⁺Cs⁺Rb⁺, Rb⁺ pulses (50 mM, 4 h) phase-shifted the rhythm and caused advances. A phase response curve was determined and the maximum steady state advances were of the order of 1 h. The dual effect of the Rb⁺ ions is discussed and is assumed to be due to two counteracting processes, exemplified by Rb⁺-sensitive ATPase-controlled pumping processes and protein synthesis. For comparison, the effects of Rb⁺ and Li⁺ in human depressive disorders is also discussed in relation to their influence on circadian systems. It is emphasized that Rb⁺ and K⁺ behave differently and are not interchangeable in their action on circadian systems.     

L'induction du rythme endogène de sporulation chez Aspergillus niger: Rõles du rapport glucose/potassium et de micro-éléments

In Aspergillus niger Van Tieghem cultivated on a synthetic medium, the induction of an endogenous rhythm of sporulation and its perpetuation depend on the glucose K⁺ balance in the medium, an excess of one of them suppressing the oscillations. In its inducing effect on the rhythm K⁺ is partially replaced by Rb⁺, but not by Na⁺, Li⁺ or Cs⁺. While the glucose K⁺ balance is favourable for the manifestation of the rhythm, the addition of increasing levels of Na⁺, Li⁺ or Cs⁺ do not modify the period length. Nevertheless, at 0.3 M of Na⁺ or Li⁺ and 0.03 M of Cs⁺ rhythm disappears. The amplitude of oscillations depends on the level of the micro-elements furnished, especially on Mn²⁺. EDTA (1 × 10^?3M) inhibits the rhythm.     

Removal by a Trace of Sodium of the Period Lengthening of the Potassium Uptake Rhythm Due to Lithium in Lemna gibba G3

The effect of Li⁺ on the period of the K⁺ uptake rhythm in the flow medium culture of the duckweed (Lemna gibba G3) was investigated under various ionic conditions. In the presence of Li⁺ at 0.2 millimolar or higher concentrations, the period was longer than the normal level of 25.4 hours by 2 hours. Li⁺ also lowered the amplitude of the rhythm. Although Na⁺ itself did not change any parameter of the rhythm, simultaneous application of Na⁺ at a very low concentration (20 micromolar) almost completely removed the effects of 0.5 millimolar Li⁺ on both the period and the amplitude. However, divalent cations (Ca²⁺ and Mg²⁺) or Rb⁺ did not remove the Li⁺ action on the period. The effect of Li⁺ and its removal by Na⁺ corresponded to intracellular Li⁺ and Na⁺ levels. The period was prolonged when the duckweed contained more Li⁺ than 5 micromoles/gram fresh weight. But the Li⁺ effect was cancelled when the in vivo Na⁺ level was greater than one-fifth that of Li⁺, even if the Li⁺ level exceeded over 5 micromoles/gram fresh weight.     

Effects of Tetraethylammoniumchloride (TEA), Vanadate,and Alkali Ions on the Lateral Leaflet Movement Rhythm of Desmodium motorium (Houtt.) Merr

The period (～3-5 min) of the ultradian rhythm of the lateral leaflet movement of Desmodium motorium is strongly lengthened (≤30-40%) by the K⁺ channel blocker tetraethylammoniumchloride (20, 30, and 40 mM) and vanadate (0.5 and 1 mM), which is an effective inhibitor of the plasma membrane-bound H⁺ pump. The alkali ions K⁺, Na⁺, Rb⁺, and Cs⁺ (10-40 mM) shorten the period only slightly (≤ 10–15%). Li⁺ (5-30 mM), however, increases the period of the leaflet rhythm drastically (≤80%). We concluded that the plasmalemma-H⁺-ATP-ase-driven K⁺ transport through K⁺ channels is an essential component of the ultradian oscillator of Desmodium, as has been proposed for the circadian oscillator.     

Potassium and Sodium Fluxes in the PhytopathogenicFungus Fusarium oxysporum var. lini

Rubidium uptake in potassium-starved cells followed biphasic kinetics in the micromolar and millimolar range and was independent of the temperature. In contrast, Rb⁺ uptake in normal-K⁺ cells followed a monophasic kinetics in the millimolar range and increased at temperatures higher than 30°C. Differences in the K _m values and in the Arrhenius plots of Rb⁺ uptake suggest different uptake systems in K⁺-starved and in normal-K⁺ cells. In addition, the substantial inhibition of Rb⁺ uptake caused by carbonyl cyanide-m-chlorophenyl hydrazone indicates that these systems are strongly dependent on membrane voltage. Lithium (sodium) tolerance, influx, and efflux were separately studied. F. oxysporum was shown to be very tolerant to sodium, while lithium caused a specific toxic effect. Li⁺ uptake in K⁺-starved cells exhibits a monophasic kinetics with low affinity. Li⁺ efflux was not affected by external pH or addition of potassium to the medium, suggesting that a Na⁺/cation antiporter is not involved in this process. Received: 14 March 2000 / Accepted: 5 June 2000     

Kinetic study on the effects of intracellular K++ and Na++ on Na++, K++, Cl+− cotransport of HeLa cells by Rb++ influx determination

Summary The effects of intracellular K⁺ and Na⁺ (K⁺ c, Na⁺ c) on the Na⁺,K⁺,Cl^+– cotransport pathway of HeLa cells were studied by measuring ouabain-insensitive, furosemide-sensitive Rb⁺ influx (JRb) at various intracellular concentrations of K⁺ and Na⁺ ([K⁺]c, [Na⁺]c). When [K⁺]c was increased and [Na⁺]c was decreased, keeping the sums of their concentrations almost constant, JRb as a function of the extracellular Rb⁺ or Na⁺ concentration ([Rb⁺]e, [Na⁺]e) was stimulated. However, the apparent K _0.5 for Rb⁺ e or Na⁺ e remained unchanged and the ratio of the apparent K ^+0.5 for K⁺ c and the apparent K _i for Na⁺ c was larger than 1. When JRb was increased by hypertonicity by addition of 200 mM mannitol, the apparent maximum JRb increased without change in the apparent K _0.5 for Rb⁺ e. These results show that K⁺ c stimulates and Na⁺ c inhibits JRb, without change in the affinities of the pathway for Rb⁺ e and Na⁺ e. The affinity for K⁺ c is slightly lower than that for Na⁺ c. Hypertonicity enhances JRb without any change in the affinity for Rb⁺ e. We derived a kinetic equation for JRb with respect to K⁺ c and Na⁺ c and proposed a general and a special model of the pathway. The special model suggests that, in HeLa cells, JRb takes place when Rb⁺ e binds to the external K⁺ binding site of the pathway after the binding of K⁺ c to the internal regulatory site.We thank Mr. T. Masuya for technical assistance. This study was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (No. 03202136) from the Japanese Ministry of Education, Science and Culture.     

Effects of ionic strength and relative humidity on the efflux of K+ (86Rb) and Ca2+ (45Ca) from roots of intact seedlings of cucumber, oat and wheat

Spring wheat (Triticum aestivum L. cv. Svenno), oat (Avena sativa L. cv. Brighton) and glasshouse cucumber (Cucumis sativus L. cv. Bestseller F1) were cultured for a week after germination on complete nutrient solutions of three different dilutions (1, 25 and 50% of the full strength medium). K⁺(⁸⁶Rb) and ⁴⁵Ca were present during the whole culture period. Relative humidity (RH) was 50% except during the last day, when half the material was transferred to 90% RH. Efflux of labelled ions was then followed during eight hours on unlabelled solutions of the same composition as before, and at both 50% and 90% RH in the atmosphere. – Uptake of K⁺(⁸⁶Rb) during growth tended to be saturated in the 25% medium. Contrariwise, the level of Ca²⁺ in the roots increased continuously with strength of the medium. At low concentrations cucumber roots were higher in Ca²⁺ than roots of oat or wheat, whereas all three species showed similar levels of Ca²⁺ in 50% medium. – At the lowest ionic strength, smooth efflux curves were obtained that could be resolved according to the three-compartment theory. At higher ionic strength, irregularities were observed, and more for Ca²⁺ than for K⁺; but for practical purposes compartment analysis with the same time constants could be applied as for the lowest concentration. – Discrimination between K⁺ and Rb⁺ differed between the roots, but not much between the shoots of different species. The roots of oat and wheat took up Rb⁺ preferentially over K⁺ in the 25% and 50% media; whereas K⁺ was preferred over Rb⁺ or little discrimination made in 1% medium and for cucumber. The shoots generally showed less discrimination than the roots. The main variability in discrimination between K⁺ and Rb⁺ thus appears to be localized in the tonoplasts of the roots cells. – Low RH around the shoots increased efflux of K⁺(⁸⁶Rb) from the cytoplasm and vacuoles of the root cells as compared to the efflux at high RH. DNP (2,4-dinitrophenol) in the medium had the same effect as high RH around the shoots. The signal system that must exist between shoots and roots is discussed as a response to “drought” conditions. In relation to investigations of others, it is assumed that the effect of DNP may indicate that part of the chain between roots and shoots consists of metabolically influenced sites, whose output is influenced by the rate of water transport.     

Mechanisms of fusicoccin action: evidence for concerted modulations of secondary K+ transport in a higher plant cell

Fusicoccin (FC) has long been known to promote K⁺ uptake in higher plant cells, including stomatal guard cells, yet the precise mechanism behind this enhancement remains uncertain. Membrane hyperpolarization, thought to arise from primary H⁺ pumping stimulated in FC, could help drive K⁺ uptake, but the extent to which FC stimulates influx and uptake frequently exceeds any reasonable estimates from Constant Field Theory based on changes in the free-running membrane potential (V _m) alone; furthermore, unidirectional flux analyses have shown that in the toxin K⁺ (⁸⁶Rb⁺) exchange plummets to 10% of the control (G.M. Clint and E.A.C. MacRobbie 1984, J. Exp. Bot.35 180–192). Thus, the activities of specific pathways for K⁺ movement across the membrane could be modified in FC. We have explored a role for K⁺ channels in mediating these fluxes in guard cells ofVicia faba L. The correspondence between FC-induced changes in chemical (⁸⁶Rb⁺) flux and in electrical current under voltage clamp was followed, using the K⁺ channel blocker tetraethylammonium chloride (TEA) to probe tracer and charge movement through K⁺-selective channels. Parallel flux and electrical measurements were carried out when cells showed little evidence of primary pump activity, thus simplifying analyses. Under these conditions, outward-directed K⁺ channel current contributed appreciably to charge balance maintainingV _m, and adding 10 mM TEA to block the current depolarized (positive-going)V _m; TEA also reduced⁸⁶Rb⁺ efflux by 68–80%. Following treatments with 10 M FC, both K⁺ channel current and⁸⁶Rb⁺ efflux decayed, irreversbly and without apparent lag, to 10%–15% of the controls and with equivalent half-times (approx. 4 min). Fusicoccin also enhanced⁸⁶Rb⁺ influx by 13.9-fold, but the influx proved largely insensitive to TEA. Overall, FC promotednet cation uptake in 0.1 mM K⁺ (Rb⁺), despite membrane potentials which were 30–60 mVpositive of the K⁺ equilibrium potential. These results tentatively link (chemical) cation efflux to charge movement through the K⁺ channels. They offer evidence of an energy-coupled mechanism for K⁺ uptake in guard cells. Finally, the data reaffirm early suspicions that FC alters profoundly the K⁺ transport capacity of the cells, independent of any changes in membrane potential.Abbreviations and symbols E _K equilibrium potential for K⁺ - FC fusicoccin - Hepes 4-(2-hydroxyethyl)-1-piperazineeth-anesulfonic acid - G _m membrane (slope) conductance atV _m - I-V current-voltage (relationship) - apparent rate constant for exchange - K _i ⁺ , K ₀ ⁺ intracellular, extracellular K⁺ (concentration) - TEA tetraethylammonium chloride - V _m free-running membrane potential (difference)     

Ability of monovalent cations to replace potassium during stimulation of lymphoid cells

Stimulation of hamster thymocytes, splenocytes, or lymph node cells occurred to a minimal extent in the absence of K⁺. This observation was found for stimulation by T-cell mitogens (phytohemagglutinin and concanavalin A), A B-cell mitogen (lipopolysaccharide), or antigen (KLH). Marginal restoration of the responses to these stimulants occurred in the presence of 0.1 mM K⁺ and responsiveness returned to near maximal levels on addition of 1 mM K⁺ to the cultures. Attempts to restore the responsiveness with other monovalent cations revealed an order of effectiveness of K⁺ ≥ Rb⁺ ? NH₄⁺ ≥ Li⁺. At the 1 mM level K⁺ and Rb⁺ were equally effective in supporting stimulation by phytohemagglutinin while all concentrations of Li⁺ tested (0.1–10 mM) would not support stimulation. However, addition of Li⁺ to cultures reconstituted with 1 mM K⁺ or Rb⁺ revealed that this ion could enhance the phytohemagglutinin response by approximately 100% in the presence of K⁺ and only 30% in the presence of Rb⁺. These data support the hypotheses that the Na,K ATPase must be active for lymphocyte stimulation to occur and that some of the biological effects of Li⁺ on lymphocyte stimulation are mediated at the level of the Na,K ATPase.     

Lithium inhibits glycogen synthase kinase-3 activity and mimics Wingless signalling in intact cells

Background Exposing eukaryotic cells to lithium ions (Li⁺) during development has marked effects on cell fate and organization. The phenotypic consequences of Li⁺ treatment on Xenopus embryos and sporulating Dictyostelium are similar to the effects of inhibition or disruption, respectively, of a highly conserved protein serine/threonine kinase, glycogen synthase kinase-3 (GSK-3). In Drosophila, the GSK-3 homologue is encoded by zw3^sgg, a segment-polarity gene involved in embryogenesis that acts downstream of wg. In higher eukaryotes, GSK-3 has been implicated in signal transduction pathways downstream of phosphoinositide 3-kinase and mitogen-activated protein kinases.Results We investigated the effect of Li⁺ on the activity of the GSK-3 family. At physiological doses, Li⁺ inhibits the activity of human GSK-3β and Drosophila Zw3^Sgg, but has no effect on other protein kinases. The effect of Li⁺ on GSK-3 is reversible in vitro. Treatment of cells with Li⁺ inhibits GSK-3-dependent phosphorylation of the microtubule-associated protein Tau. Li⁺ treatment of Drosophila S2 cells and rat PC12 cells induces accumulation of cytoplasmic Armadillo/β-catenin, demonstrating that Li⁺ can mimic Wingless signalling in intact cells, consistent with its inhibition of GSK-3.Conclusions Li⁺ acts as a specific inhibitor of the GSK-3 family of protein kinases in vitro and in intact cells, and mimics Wingless signalling. This reveals a possible molecular mechanism of Li⁺ action on development and differentiation.     

Effects of lithium salts on the behaviour and the orcadian system of Mesocricetus auratus W.

Abstract

LiCl (195 μg/g body weight and day) reduces water uptake in Syrian hamsters by 40%. Sleep duration is increased from 50% per day to 60% per day. Other behavioural items are hardly influenced. Shortening and lengthening of the circadian period was induced by Li⁺ in individual hamsters, but the mean period of the population was not changed. The upper limit of entrainment is increased by Li⁺.     

Effects of monovalent cations on the activities associated with coupling site III of rat liver mitochondria

The effects of substitution of K⁺ by Li⁺, Na⁺, or Rb⁺ in the assay medium on the processes of electron transfer and H⁺ translocation associated with Site III are investigated. The replacement of K⁺ with Rb⁺ has little effect, if any, on the measured initial rates of H⁺ extrusion and electron transfer. The substitution of K⁺ by Li⁺ increases the initial rate of both processes simultaneously while the replacement of K⁺ by Na⁺ causes an enhancement on the rate of electron transfer with concomitant inhibition of the observed acidification. The presence of either Na⁺ or Li⁺ decreases the proton-leak rate of the inner membrane. These results suggest that the link between electron transfer and H⁺ translocation in Site III is weakened by the presence of Na⁺.     

Sodium/potassium selectivity and pleiotropy in stl2, a highly salt-tolerant mutation of Ceratopteris richardii

The roles of Na⁺ and K⁺ (Rb⁺) uptake were further studied in a NaCl-tolerant strain of Ceratopteris richardii containing the stl2 mutation by direct comparison with the wild-type strain. In addition to Na⁺ tolerance, stl2 also confers tolerance to Mg²⁺ and sensitivity to K⁺. In addition to higher K⁺ (Rb⁺) uptake at concentrations commonly associated with low-affinity K⁺ transport, stl2 maintained higher uptake down to 0·1 mol m^–3 Rb⁺. Up to a 25-fold excess of Na⁺ had little effect in either genotype on K⁺ (Rb⁺) uptake at low concentrations, i.e. 0·2 and 0·5 mol m^–3 RbCl. Pretreatment with K⁺ (20 mol m^–3) inhibited uptake of K⁺ (Rb⁺) in the wild type, whereas concurrent inclusion of K⁺ inhibited uptake of Rb⁺ more in stl2. In the absence of K⁺, Na⁺ uptake (0·01–60 mol m^–3) was nearly identical in the wild type and stl2. K⁺ inhibited Na⁺ uptake more effectively in stl2 than the wild type, especially at 60 mol m^–3 Na⁺. Greater inhibition of K⁺ uptake in stl2 occurred with MgCl₂ or TEA (tetraethylammonium chloride) preincubation or with simultaneous inclusion of Al³⁺ (Al₂SO₄). The higher effective velocity of K⁺ uptake at a wide range of concentrations and the enhanced selectivity for K⁺ and against Na⁺ contribute to the preservation of higher cytosolic K⁺ and lower Na⁺ under salinity stress.     

Effects of monovalent cations on phosphate accumulation and storage of the ectomycorrhizal fungus Suillus bovinus

Comparative in vivo ³¹P-NMR studies of the fungus Suillus bovinus (L.: Fr.) O. Kuntze in pure culture have produced interesting new data. To investigate the response of phosphate metabolism to a change in external monovalent cations, samples were exposed to a Hoagland solution containing different monovalent cations Li⁺, Na⁺, K⁺, or Rb⁺ at 10 mM concentration. A method of nutrient cycling during analysis where the cation was changed and the phosphate kept constant allowed us to determine the kinetics of phosphate accumulation, storage and incorporation into polyphosphate following exposure to the range of test cations. Different external monovalent cations had different effects upon changes in the content of both phosphate and polyphosphate. Treatment with Li⁺, Na⁺, or Rb⁺ resulted in a change in phosphate accumulation to 60, 73, and 107% and in content of the intracellular mobile polyphosphate (polyP) to 119, 112, and 94%, respectively, compared with the control taken as 100%. The effect of each cation is related to its position in the periodic table. Reversing this process, i.e., exchanging with K⁺, returned phosphate metabolism to normal. Although, the increase in depolarization of the cell membrane should affect the internal pH, fungal metabolism using energy requiring mechanisms appeared necessary to maintain the intracellular pH. Thus, increasing contents of mobile polyP were the consequence of an increasing energy demand. On the other hand, the increasing depolarization of the cell membrane following the sequence Rb⁺ < K⁺ < Na⁺ < Li⁺ inhibited the net P_i accumulation. Furthermore, it is postulated that the P_i accumulation was also regulated by the intracellular content in polyP.     

Effect of pH and Sodium Ion on Germination of Alkalophilic Bacillus Species

The factors affecting the germination of spores of alkalophilic Bacillus species have been studied. The optimum pH for germination of spores of alkalophilic Bacillus No. 2b-2 was about 10 and NaCl (Na⁺) stimulated germination considerably. The optimum concentration of NaCl for germination was about 0.2 M. Other cations such as K⁺, NH₄⁺, Rb ⁺ , Cs⁺ and Ca²⁺ did not stimulate germination. Li⁺ showed a weak activity for stimulating germination. Na⁺ ions stimulated the early step of germination. It was necessary for Na⁺ ions to co-exsist with the germinants in the germination of spores and the effect of Na⁺ was reversible. The same results were obtained for the germination of alkalophilic Bacillus No. 16-2 and No. 20.     

Nigericin forms highly stable complexes with lithium and cesium

Nigericin is a monocarboxylic polyether molecule described as a mobile K⁺ ionophore unable to transport Li⁺ and Cs⁺ across natural or artificial membranes. This paper shows that the ion carrier molecule forms complexes of equivalent energy demands with Li⁺, Cs⁺, Na⁺, Rb⁺, and K⁺. This is in accordance with the similar values of the complex stability constants obtained from nigericin with the five alkali metal cations assayed. On the other hand, nigericinalkali metal cation binding isotherms show faster rates for Li⁺ and Cs⁺ than for Na⁺, K⁺, and Rb⁺, in conditions where the carboxylic proton does not dissociate. Furthermore, proton NMR spectra of nigericin-Li⁺ and nigericin-Cs⁺ complexes show wide broadenings, suggesting strong cation interaction with the ionophore; in contrast, the complexes with Na⁺, K⁺, and Rb⁺ show only clear-cut chemical shifts. These latter results support the view that nigericin forms highly stable complexes with Li⁺ and Cs⁺ and contribute to the explanation for the inability of this ionophore to transport the former cations in conditions where it catalyzes a fast transport of K⁺>Rb⁺>Na⁺.Part of the results of this paper were presented at the 14th International Congress of Biochemistry in Prague, Czechoslovakia.     

Effects of monovalent cations on red cell shape and size

Human erythrocytes were incubated in isotonic solutions of different monovalent cations. The apparent size of the red cells measured on scanning electron microscopic pictures decreases in the order Li⁺>Na⁺=K⁺>Rb⁺. These differences in size are abolished after pretreatment with trypsin, which removes a large part of the charges associated with membrane glycoproteins. Shape alterations are also observed. Normal biconcave shapes are visible after Na⁺ or K⁺ incubation, whereas Li⁺ leads to flabby, flattened cells with a certain tendency to crenation, and Rb⁺ causes more pronounced biconcavity with a certain tendency to cupping. The overall effects of pretreatment with trypsin are similar to those of Li⁺. Our results provide evidence that the electrostatic repulsion of glycoproteins and other charged membrane components may play an essential role in maintaining red cell shape.     

Potassium transport in Chlamydomonas reinhardtii: isolation and characterization of transport-deficient mutant strains

Putative potassium-transport-deficient mutant strains of Chlamydomonas reinhardtii Dang. were induced by ultra-violet mutagenesis and were identified by their dependence on abnormally high concentrations of potassium for growth. Potassium transport studies employing ⁸⁶Rb as a tracer were carried out with wild-type cells and with three independently isolated KDP (potassium-dependent phenotype) clones. Wildtype cells exhibit two transport activities. Transport activity A was expressed when cells were grown in medium supplemented with 10 mM KCl. The transporter with type-A activity does not discriminate between either Rb⁺ or K⁺ as a substrate and has a K_m for Rb⁺ equal to 1 mM and a V_max equal to 31 nmol Rb⁺ h^-1 10^-6 cells. Transport activity B was expressed when cells were starved of potassium for 24 h. The transporter with type-B activity prefers K⁺ to Rb⁺ as a substrate; it has a K_m for Rb⁺ equal to 2.5 mM and a V_max equal to 210 nmol Rb⁺ h^-1 10^-6 cells. All three mutant clones exhibit transport activity comparable to type-A when grown in 10 mM KCl. When starved of potassium for 24 h, two KDP clones demonstrate no transport activity and the third clone continues to exhibit only type-A activity.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DES diethylstilbesterol - KDP potassium-dependent phenotype     

Thiol-Dependent passive K/Cl transport in sheep red cells: IV. Furosemide inhibition as a function of external Rb+, Na+, and Cl−

Summary The effect of the loop diuretic furosemide (4-chloro-N-furfuryl-5-sulfamoyl-anthranilic acid) on the thiol-dependent, ouabain-insensitive K(Rb)/Cl transport in low K⁺ sheep red cells was studied at various concentrations of extracellular Rb⁺, Na⁺ and Cl^–. In Rb⁺-free NaCl media, 2×10^–3 m furosemide inhibited only one-half of thiol-dependent K⁺ efflux. In the presence of 23mm RbCl, however, the concentration of furosemide to produce 50% K⁺ efflux inhibition (IC₅₀) was 5×10^–5 m. In Rb⁺ containing NaCl media, the inhibitory effect of 10^–3 m furosemide was equal to that caused by NO ₃ ^– replacement of Cl^– in the medium. The apparent synergistic action of furosemide and external Rb⁺ on K⁺ efflux was also seen in the ouabain-insensitive Rb⁺ influx. A preliminary kinetic analysis suggests that furosemide binding alters both maximal K⁺(Rb⁺) transport and apparent external Rb⁺ affinity. In the presence of external Rb⁺, Na⁺ (as compared to choline) exerted a small but significant augmentation of the furosemide inhibition of K⁺(Rb⁺) fluxes. There was no effect of Cl^– on the IC₅₀ value of furosemide. As there is no evidence for coupled Na⁺K⁺ cotransport in low K⁺ sheep red cells, furosemide may modify thiol-dependent K⁺(Rb⁺/Cl flux or Rb⁺ (and to a slight degree Na⁺) modulate the effect of furosemide.     

The effect of monovalent cations on the association behavior of guanosine 5'-monophosphate, cytidine 5'-monophosphate, and their equimolar mixture in aqueous solution

¹H- and ³¹P-nmr spectroscopy have been used to investigate the self-association of M₂(5′-CMP) [M = Li⁺, Na⁺, K⁺, Rb⁺, or (CH₃)₄ N⁺; 5′-CMP = cytidine 5′-monophosphate], the self-association of Li₂(5′-GMP) (5′-GMP = guanosine 5′-monophosphate), and the heteroassociation of 5′-GMP and 5′-CMP (1 : 1 mole ratio) in aqueous solution as a function of the nature of the monovalent cation. Proton spectral differences for the different 5′-CMP salts exhibit a cation-size dependence and have been ascribed to a change in the stacking geometry. An average stacking association constant of 0.63 ± 0.24M^?1 at 1°C, consistent with the weak stacking interactions of the cytosine bases, was determined for the 5′-CMP salts. Heteroassociation of 5′-GMP and 5′-CMP follows the reverse of the cation order for the formation of ordered aggregates of 5′-GMP. Heteroassociation occurs in the presence of Li⁺, Na⁺, and Rb⁺ ions, but only self-association occurs for the K⁺ nucleotides. Li₂(5′-GMP), which does not form ordered species, self-associates to form disordered base stacks with a stacking constant of 1.63 ± 0.11M^?1 at 1°C.