Regulation of Na+ transport in brown adipose tissue.

In order to test the hypothesis that Na+, K+-ATPase (Na+,K+-dependent ATPase) is involved in the noradrenaline-mediated stimulation of respiration in brown adipose tissue, the effects of noradrenaline on Na+,K+-ATPase in isolated brown-fat-cell membrane vesicles, and on 22Na+ and K+ (86Rb+) fluxes across the membranes of intact isolated cells, were measured. The ouabain-sensitive fraction of the K+-dependent ATPase activity in the isolated membrane-vesicle preparation was small and was not affected by the presence of noradrenaline in the incubation media. The uptake of 86Rb+ into intact hormone-sensitive cells was inhibited by 80% by ouabain, but it was insensitive to the presence of noradrenaline. 22Na+ uptake and efflux measured in the intact cells were 8 times more rapid than the 86Rb+ fluxes and were unaffected by ouabain. This indicated the presence of a separate, more active, transport system for Na+ than the Na+,K+-ATPase. This is likely to be a Na+/Na+ exchange activity under normal aerobic conditions. However, under anaerobic conditions, or conditions simulating anaerobiosis (2 mM-NaCN), the unidirectional uptake of Na+ increased dramatically, while efflux was unaltered.     

Cell-cell communication in the leaves of Commelina cyanea and other plants

Abstract. The fluorescent probes 6–carboxyfluorescein and lucifer yellow CH which do not pass the plasmalemma have been used to examine cell-to-cell communication in the leaf of Commelina cyanea. Dye movement from cell-to-cell occurs in epidermal, spongy and palisade mesophyll, and vascular cells. Dye movement between these tissues was also found. Hence, the epidermis, spongy and palisade mesophyll cells, and vascular tissue are all linked in a continuous symplast. However, dye injected into the epidermal cells rarely moves into guard cells, indicating that these cells are relatively isolated from the surrounding cells. In the same way, guard cells in Vicia faba and the C₄ grass Anthephora pubescens also appeared to be isolated from epidermal cells. Thus guard cell isolation from cell-to-cell communication appears to be a common phenomenon. Hence, the ion fluxes required for guard cell function must occur via the apoplast.     

Energetics of 22Na+ Absorption by Bean-leaf Slices

Sodium absorption by bean-leaf slices from o·1 mM ²²NaCldepends on metabolic energy, but is not enhanced by light. Lightalso does not effect the ATP content of the tissue. Inhibitorsof energy metabolism which decrease the ATP content also inhibit²²Na⁺ absorption. In darkness, anoxia severely affects the ATPcontent as well as ²²Na⁺ absorption; illumination restores bothto almost normal values.     

Effect of calcium channel blockers on serotonin uptake

We previously demonstrated that verapamil inhibits serotonin uptake by bovine pulmonary arterial endothelial cells by a mechanism not involving alterations in calcium fluxes. In this study, we determine whether verapamil inhibition of serotonin uptake occurs in other pulmonary cell types (bovine pulmonary artery smooth muscle cells), in cells from other organs and species (rat epididymal endothelial cells), and in intact organs (isolated rat lungs). We also compare the effects of verapamil with those of nifedipine and diltiazem. At concentrations of 10(-6) M or greater, verapamil is an inhibitor of serotonin uptake by cultured cells and isolated lungs. Nifedipine and diltiazem are weak inhibitors of serotonin uptake by cultured bovine cells only at suprapharmacologic doses and have no effect on serotonin uptake by isolated lungs. Surprisingly, nifedipine stimulates serotonin uptake by rat epididymal endothelial cells. We conclude that inhibition of serotonin uptake by verapamil is a generalized phenomenon, occurring in a variety of cell types, in intact organs, and in different species that does not occur consistently with other calcium channel blockers.     

Melittin stimulates Na entry,Na-K pump activity and DNA synthesis in quiescent cultures of mouse cells

Melittin, an amphipatic polypeptide, increases several fold the activity of Na-K pump in quiescent Swiss 3T3 cells. As with other growth factors, melittin increases the activity of the pump by increasing Na entry into the cell. In contrast, other early responses are not elicited by the toxin. At concentrations that promote ion fluxes, melittin stimulates DNA synthesis in quiescent mouse cells acting synergistically with insulin, epidermal growth factor and with the growth factor released by SV40 BHK cells. In contrast, melittin does not interact synergistically with either phorbol esters or vasopressin. The cellular effects of melittin are consistent with the proposal that ion fluxes signal the initiation of mitogenesis in quiescent cells.     

Potassium transport by rabbit descending colon, in vitro

Studies of the bidirectional fluxes of K across segments of rabbit descending colon indicate that: a) when the tissue is short-circuited, the net flux does not differ significantly from zero under control conditions and in the presence of aldosterone; and b) the bidirectional fluxes of K conform to the Ussing flux-ratio equation over a wide range of transepithelial electrochemical potential differences. These and other findings strongly suggest that the movements of K across the epithelium are restricted to paracellular routes and are entirely passive. Studies dealing with the mechanism of homocellular K transport indicate that: a) K is actively transported into the cells across the basolateral membranes against an electrochemical potential difference of approximately 30 mV; and b) the active uptake of K may be mediated by a rheogenic Na-K exchange pump that is also responsible for transcellular Na transport. These results are entirely consistent with the model proposed by Koefoed-Johnson and Ussing for isolated frog skin.     

Chloride movements in human neutrophils. Diffusion, exchange, and active transport

Chloride content and fluxes were measured in isolated resting human peripheral polymorphonuclear leukocytes. The intracellular Cl concentration of cells kept at 37 degrees C in 148 mM Cl media was approximately 80 meq/liter cell water, fourfold higher than expected for passive distribution at the cell's estimated membrane potential (approximately -53 mV). All intracellular Cl was rapidly exchangeable with external 36Cl. Cells lost Cl exponentially into Cl-free media, and reaccumulated it when Cl was restored to the bath; this reuptake was dependent on metabolism. One-way 36Cl fluxes in steady state cells were approximately 1.4 meq/liter X min. The bulk (approximately 70%) of these represented electrically silent Cl/Cl exchange mediated by a carrier insensitive to disulfonic stilbenes but blocked by the anion carrier inhibitor alpha-cyano-4-hydroxycinnamate (CHC). The remaining fluxes were characterized in some detail. About 20% of 36Cl influx behaved as active transport: it moved thermodynamically uphill and was absent in cells treated with 2-deoxy-D-glucose, displayed Michaelis-Menten kinetics with Km(Cl) congruent to 5 mM, Vmax congruent to 0.25 meq/liter X min, and was inhibited by CHC (Ki congruent to 1.7 mM), ethacrynate (Ki congruent to 50 microM), and furosemide (Ki congruent to 50 microM). About 30% of Cl efflux and approximately 8% of Cl influx behaved as electrodiffusion through a low-permeability pathway (PCl congruent to 4 X 10(-9) cm/s; gCl congruent to 1 microsecond/cm2; PK/PNa/PCl congruent to to 10:1:1); these fluxes were linear with concentration and strongly voltage sensitive. The putative Cl channel does not appear to be voltage gated, and gives evidence of single filing.     

Sodium Fluxes in the Erythrocytes of Swine, Ox, and Dog

Sodium fluxes were measured in erythrocytes from three species of mammals. Unidirectional fluxes were slowest in swine RBCs (low sodium cells), fastest in dog RBCs (high sodium cells), and between these extremes in ox cells (intermediate level of internal sodium). In addition, efflux and influx in swine cells both correlated positively with intracellular sodium concentration between 12 to 4 µeq/ml. Tracer effluxes in swine and beef cells were separated into three components: active transport, diffusion, and exchange diffusion. The last two also contributed to influx. Transport was greater in swine cells than in beef, while the leak was similar in both. Pump to leak ratios were about 21 for swine and 3 for beef, a difference that probably accounts for the higher cell sodium in the latter. Exchange diffusion was faster in beef cells than in swine resulting in a larger tracer movement in beef. The exchange mechanism was temperature-sensitive, but was not inhibited by strophanthin. The unidirectional fluxes in canine cells were inhibited by low temperature, but they were sensibly unaffected by strophanthin. When placed in magnesium Ringer's solution (inhibits exchange diffusion in beef and swine cells) dog RBCs lost more than half of their internal sodium at a rate approximating the isotope flux in plasma or normal Ringer's solution. It was, however, not possible to separate the total tracer movement into pump, leak, and exchange.     

Sodium and potassium fluxes and membrane potential of human neutrophils: evidence for an electrogenic sodium pump

Sodium and potassium ion contents and fluxes of isolated resting human peripheral polymorphonuclear leukocytes were measured. In cells kept at 37 degrees C, [Na]i was 25 mM and [K]i was 120 mM; both ions were completely exchangeable with extracellular isotopes. One-way Na and K fluxes, measured with 22Na and 42K, were all approximately 0.9 meq/liter cell water . min. Ouabain had no effect on Na influx or K efflux, but inhibited 95 +/- 7% of Na efflux and 63% of K influx. Cells kept at 0 degree C gained sodium in exchange for potassium ([Na]i nearly tripled in 3 h); upon rewarming, ouabain-sensitive K influx into such cells was strongly enhanced. External K stimulated Na efflux (Km approximately 1.5 mM in 140-mM Na medium). The PNa/PK permeability ratio, estimated from ouabain insensitive fluxes, was 0.10. Valinomycin (1 microM) approximately doubled PK. Membrane potential (Vm) was estimated using the potentiometric indicator diS-C3(5); calibration was based on the assumption of constant-field behavior. External K, but not Cl, affected Vm. Ouabain caused a depolarization whose magnitude dependent on [Na]i. Sodium-depleted cells became hyperpolarized when exposed to the neutral exchange carrier monensin; this hyperpolarization was abolished by ouabain. We conclude that the sodium pump of human peripheral neutrophils is electrogenic, and that the size of the pump-induced hyperpolarization is consistent with the membrane conductance (3.7-4.0 microseconds/cm2) computed from the individual K and Na conductances.     

Deoxygenation-induced cation fluxes in sickle cells: relationship between net potassium efflux and net sodium influx

Deoxygenation-induced cation fluxes in sickle cells were studied by measuring net cation movements in ouabain-treated cells. These deoxy cation fluxes were highly dependent on pH, showing inhibition at pH less than 7 and greater than 8 and a maximum at 7.4-7.5. Activation occurred at oxygen tensions around 40-50 torr and fluxes rose sharply as PO2 fell lower. Deoxy K efflux paralleled deoxy Na influx at pH values between 7 and 8, and at all oxygen tensions. Sickle cells were separated by density on Percol-Stractan gradients. Dense cells had lower deoxy cation fluxes of both Na and K than did lighter cell fractions, but in none of the fractionated populations did deoxy K efflux exceed deoxy Na influx. These data demonstrate that deoxy cation fluxes are activated at physiological pH and oxygen tensions and that there are no conditions of pH and PO2 and no cell populations in which cation fluxes induced by deoxygenation contribute directly to net cation loss in sickle cells. Chloride replacement (with nitrate) did not alter deoxy cation fluxes, and deoxy K efflux did not require the presence of external Na (tetramethylammonium replacement). Thus, deoxy cation fluxes do not have the characteristics of a cation-chloride cotransport or cation countertransport system.     

Measurements of net fluxes and extracellular changes of H+, Ca2+, K+, and NH4+ in Escherichia coli using ion-selective microelectrodes.

This study introduced the use of a non-invasive ion-selective microelectrode (MIFE) technique to study membrane-transport processes in bacteria. Net ion fluxes and changes in the extracellular concentrations of H+, Ca2+, K+ and NH4+ in adherent bacteria, isolated from cultures at different growth stages (exponential, late exponential, and stationary phases), were monitored. With the exception of Ca2+, a significant (P=0.05) difference was found in the magnitude of net fluxes of the ions measured from bacterial cells at different stages of the population growth curve. The magnitude of the H+ response was glucose-dependent with maximum changes occurring at the highest concentration. There was a progressive increase in H+ extrusion followed by a gradual return to zero at late stationary phase. Measurements of net ion fluxes crossing the bacterial cytoplasmic membrane, demonstrated here for the first time, may offer insight into underlying mechanisms of ion transport kinetics. Applications of the non-invasive ion-selective microelectrode technique in microbiology are discussed.     

General Mechanisms for Solute Transport Across the Tonoplast of Plant Vacuoles: a Patch-Clamp Survey of Ion Channels and Proton Pumps

The electrical properties of the tonoplast from a large variety of plant materials such as mesophyll cells, storage cells, tumor cells, suspension cultured cells, guard cells, coleoptile cells, and liverwort cells have been investigated using the patch-clamp technique. Whole-vacuole recordings were employed to study the dynamics of an ATP-dependent proton pump by directly measuring the electrogenic currents. The addition of Mg-ATP induced an inwardly directed current which depolarized the tonoplast (the vacuole becoming positive inside). Furthermore, voltage-dependent passive ion fluxes were analyzed using whole vacuoles and isolated membrane patches. Whole-vacuolar currents and single-channel currents were induced at hyperpolarizing potentials, whereas currents decreased at positive trans-tonoplast potentials. The electrical properties of the tonoplast of vacuoles from various plant tissues were similar and it was concluded that ion fluxes across the tonoplast follow the same general mechanisms.     

Effects of NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) on chloride transport in intestinal tissues and the T84 cell line.

NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) has been reported to block Cl- channels in isolated rabbit nephrons with high potency (IC50 = 80 nM). The effects of this compound on Cl(-)-mediated transport processes in intestinal tissues have been studied using agonist-stimulated short-circuit current (T84) in Ussing chamber experiments and 36Cl- fluxes in monolayers of a colonic cell line (T84). NPPB inhibited PGE1-stimulated Isc in rabbit distal colon and ileum at concentrations in the range 20 to 100 microM. However, NPPB at the same concentrations also inhibited glucose-stimulated Isc in rabbit ileum, suggesting that its effects were not restricted to those on Cl- transport. Consistent with this, exposure of rabbit distal colon to 100 microM NPPB was found to reduce endogenous ATP levels by 69%, implying that, at these concentrations, NPPB could impair active transport processes by an effect on cellular energy metabolism. Clear evidence for a direct effect of NPPB on epithelial chloride channels was found in studies on Cl- fluxes in T84 cell monolayers. NPPB inhibited VIP-stimulated Cl- uptake into T84 cells with an IC50 of 414 microM. NPPB (1 mM) also inhibited Cl- efflux from pre-loaded cells confirming its effect as a weak Cl- channel blocker in this system.     

Effects of nicorandil on isolated smooth muscle cells from guinea-pig taenia caeci

1. The effects of nicorandil on guinea-pig taenia caeci were investigated with the use of isolated smooth muscle cells and glycerin-treated muscle fiber bundles. 2. Nicorandil inhibited high K-, Ca2+- and carbachol-induced contractions in a dose-dependent manner without affecting 45Ca fluxes in isolated cells. 3. Nicorandil had no effect on ATP-induced contraction of glycerin-treated muscle fiber bundles. 4. The present results suggest that nicorandil may inhibit the contraction by action on the contractile proteins in an indirect manner in guinea-pig taenia caeci.     

Unidirectional ion movements in the hindgut of larval Sarcophaga bullata (Diptera: Sarcophagidae).

1. Unidirectional Na+, K+, and Cl- fluxes were measured across the isolated hindgut of larval Sarcophaga bullata. 2. Both K+ and Cl- are actively secreted into the hindgut lumen, whereas Na+ is distributed passively. 3. The movements of K+ and Cl- are not entirely independent of each other, and the movement of one ion influences the flux of the co-ion. 4. The NH4+ ion is secreted into the hindgut by a mechanism separate from K+ secretion.     

Development of hormonal regulation of ion transport in embryonic chick red cells

We have found that cation transport in red cells from chick embryos is stimulated by the hormone epinephrine and that this response develops as the embryonic definitive cells mature. Sodium efflux and potassium influx are significantly stimulated (50%) by epinephrine in red cells from embryos incubated ten days or longer, whereas cation fluxes in erythroid cells from 8- or 9-day embryos are stimulated little or not at all. The effect of epinephrine may be mediated by cyclic AMP as adenylate cyclase activity in membranes isolated from embryonic red cells is only slightly stimulated at nine days, but the response increases as the cells mature to a maximum of about 180%. Also the stimulation of cation transport by epinephrine is blocked by propranolol, but not by phentolamine. Although the younger cells respond poorly to epinephrine, cyclic AMP significantly stimulates transport. The enhancement of cation fluxes by epinephrine or cyclic AMP occurs even in the presence of ouabain. Since both K influx and Na efflux are enhanced by these agents, their action is most likely on some form of the “Na-K” pump which is not ouabain sensitive resulting in a significant increase in the maximum velocity of the pump. We suggest the hypothesis that there are two classes of “Na-K” pump in these embryonic cells. One pump is similar to that found in many erythrocytes including mammalian cells in that it selectively pumps potassium in and sodium out, is ouabain-sensitive, and is primarily involved in maintaining intracellular cation concentrations. The second pump is enhanced by epinephrine via cyclic AMP, is not inhibited by ouabain, and may have lower ion selectivity. This hormone sensitive pump activity is lost as the cells mature, a process which is completed when the animal is fully grown and no longer has significant numbers of embryonic cells in its circulation.     

The Effects of Fusicoccin on Anion Fluxes in Isolated Guard Cells of Commelina communis L.

Clint, G. M. 1987. The effects of fusicoccin on anion fluxesin isolated guard cells of Commelina communis L.—J. exp.BoL 38: 863–876. The effects of 3?10^–2 mol m^–3 fusicoccin (FC) onbromide fluxes and contents in isolated guard cells of Commelinacommunis L. have been studied using K⁸²Br at pH 3?9 and pH 6?7.At pH 3?9 FC caused a reduction in both the influx and the effluxof ⁸²Br, whereas at pH 6?7 FC had no effect on the influx butcaused a transient increase in the efflux of ⁸²Br. There wasno obvious change in bromide content with FC treatment at eitherpH. The behaviour of the anion fluxes in response to FC suggeststhat FC does not act solely via a hyperpolarization at the plasmalemma.A redistribution of bromide between the intracellular compartmentssuggests that anion flux from the cytoplasm to the vacuole maybe stimulated by FC at pH 3?9. The failure of guard cells toincrease their anion content on treatment with FC despite anincrease in stomatal aperture and in cation content suggeststhat in FC-induced stomatal opening excess cation is balancedby organic acid synthesis within the guard cell. Key words: Fusicoccin, guard cells, ion fluxes, Commelina communis     

Do microbial numbers count? Quantifying the regulation of biogeochemical fluxes by population size and cellular activity

In order to enhance understanding of the interrelationships among community members and between them and their environment, the concept of regulation analysis is extended from biochemistry into microbial ecology. Ecological regulation analysis quantifies how biogeochemical fluxes are regulated by the microorganisms performing the process; the degree to which changes in fluxes are due to changes in population size and to changes in activity cell(-1) (cellular activity). Regulation analysis requires data on biogeochemical fluxes and the numbers of cells through which these fluxes run. Its application to five biogeochemical processes (aerobic methane oxidation, aerobic nitrite oxidation, methanogenesis, sulfate reduction and reductive dehalogenation) revealed that in general, but not always, flux was primarily regulated by cellular activity, i.e. by changes in the size and properties of the enzyme pool and in the concentrations of substrates and metabolites. Thus, it is often not sufficient to count the numbers of cells performing a particular step in a biogeochemical process in order to estimate its flux. Ecological regulation analysis can be extended to address which aspects of cellular activity require quantification in order to describe biogeochemical fluxes better. Its application is discussed in the context of the complexity of microbial communities (e.g. functional redundancy) and their functioning.     

Compartmentation and fluxes of inorganic phosphate in photosynthetic cells

An analysis of the compartmentation and fluxes of inorganic phosphate in isolated cladophyll cells from Asparagus officinalis was made in parallel with an ultrastructural study. The elution pattern of labelled inorganic phosphate (which indicates that the asparagus cells are behaving as a system of three compartments in series) was used to quantify the fluxes between the vacuole, cytoplasm and free space. A relaxation time of 198 min was calculated for inorganic phosphate exchange between the vacuole and cytoplasm. It is, therefore, suggested that the vacuole serves to buffer the cytoplasmic inorganic phosphate concentration in the long term. However, in the short term, exchange with the vacuole will not appreciably affect the cytoplasmic inorganic phosphate concentration and thus the partitioning of photosynthetically fixed carbon.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - P_i inorganic phosphate     

Ionic fluxes in isolated epithelial cells of the abdominal skin of the frog Leptodactylus ocellatus.

Unidirectional ion fluxes are measured in cells isolated by a trypsination-dissection method from the epithelium of the frog Leptodactylus ocellatus. Potassium seems to be contained in a single cellular compartment. The influx of potassium is 0.0068 mumole min-1 mg-1 of dry weight and is carried by a ouabain-sensitive pump. Sodium seems to be contained in two cellular compartments, one of which does not exchange its Na within the experimental period. The possibility that these compartments reflect the existence of different types of cells is not discarded. 49% of the rate constant for the Na efflux is ouabain-sensitive and 23% is ethacrynic-sensitive. Under control conditions the permeability to potassium (PK), sodium (PNa) and chloride (PC1) are 7.6 X 10(-5), 2.6 X 10(-5) and 2.8 X 10(-5) liters/min mg, respectively. The value of PNa is much higher than predicted by current electrical models of the epithelium. The discrepancy might offer some insight into the nature of the "inner facing barrier" of the skin.