Influx Measurements Research Articles

Direct measurement of sodium influx in vascular smooth muscle. Stimulation by aldosterone.

The influx of 22Na into the smooth muscle cells of incubated rat tail arteries was measured directly, in the presence and absence of 2.8 X 10(-7) M D-aldosterone. During the analysis, extracellular 22Na and 23Na were removed by incubation of the tissue in sodium-free lithium-substituted physiological salt solution at less than 3 degrees C. Aldosterone increased the influx rate coefficient by 10% (0.104 +/- 0.004 [SE] min-1, n = 13, vs 0.095 +/- 0.003 [SE] min-1, n = 13 without aldosterone; p less than 0.01) but did not significantly reduce the sodium content. We conclude from direct measurements that aldosterone acts to increase the influx of sodium in incubated tail arteries from normal rats; we conclude from measurements of the sodium influx and sodium content that aldosterone also acts to increase the efflux of sodium in this preparation.

Measurement of calcium influx in tethered rings of rabbit aorta under tension.

Calcium (Ca) influx in vascular smooth muscle is routinely measured in untethered preparations not under passive stretch, and Ca influx data are correlated with data for steady-state isometric tension obtained under parallel conditions from tethered preparations under passive stretch. The validity of this method was tested by simultaneous measurement of Ca influx and tension in tethered rings of rabbit thoracic aorta. Ca influx (45Ca 3-min pulse) and tension were measured at 3 and 30 min after norepinephrine (NE) or KCl and under control (no agonist) conditions. Active tension was significantly altered by variations in passive tension. Ca influx was unaffected by passive tension under control, NE, or KCl conditions, and results were similar at 3 and 30 min. The results confirm the validity of correlating Ca influx data from untethered rings with steady-state contractile response data obtained from tethered rings under similar experimental conditions.

Sodium Balance in Fresh-Run Atlantic Salmon

Sodium Balance in Fresh-Run Atlantic Salmon

Effect of ethanol on choline transport in rat jejunum.

The effect of ethanol on choline transport across the rat jejunum was studied by intraluminal perfusion in vivo and by influx measurement across the brush-border membrane in vitro. Acute ethanol administration (4 g/kg) through a gastric tube caused an increase in net choline absorption within 1 h. The increase was prevented by pretreatment with pyrazole, an inhibitor of ethanol metabolism. Chronic ethanol administration also caused an increase in choline absorption, the effect being unrelated to the nutritional changes that occur with ethanol ingestion. In contrast, direct instillation of 0.65 M ethanol through the perfusate caused no changes in choline absorption, and the perfusion of a 1.14 M solution even decreased absorption. The in vitro influx of choline across the mucosal membrane of the isolated rat jejunum was also enhanced by pretreatment with ethanol given by gavage 1 h prior to experimentation. Similarly, the ethanol-related increase in the influx rate was inhibited by pyrazole but was unaffected by acetaldehyde or acetate. Like ethanol, pretreatment of rats with methanol stimulated the choline influx rate. The results suggest that ethanol metabolism, rather than the direct effect of ethanol by itself, stimulates the absorption and influx of choline into the rat jejunum. The effect is not produced by the primary metabolites of ethanol, acetaldehyde, or acetate but is very likely related to stimulation by other products of ethanol metabolism.

A quenched-flow technique for the measurement of glucose influx into human red blood cells

A quenched-flow apparatus is described and applied to measurements of the hydrolysis of 2,4-dinitrophenyl acetate by sodium hydroxide and the entry of d-[U- 14C]glucose into human red blood cells at 37°C. Glucose influx into red cells was a saturable process obeying Michaelis-Menten kinetics with a K m for glucose of 6.6 ± 0.61 m m and a maximum rate for glucose entry under “zero trans” conditions of 20.7 ± 0.76 mmol (L cell water) −1 s −1. The technique used requires only readily available laboratory equipment and should be easily adaptable to the study of other rapid transport processes.

Interaction of brevetoxin A with a new receptor site on the sodium channel

W. A. Catterall and M. Gainer. Interaction of brevetoxin A with a receptor site on the sodium channel. Toxicon 23, 497 – 504, 1985. — Measurements of neurotoxin-activated 22Na influx in neuroblastoma cells and neurotoxin binding in synaptosomes were used to define the site and mechanism of action of brevetoxins on sodium channels. Brevetoxin A alone did not enhance the sodium permeability of neuroblastoma cells, but markedly enhanced persistent activation of sodium channels by veratridine, aconitine and batrachotoxin, which act at neurotoxin receptor site 2. Enhancement of batrachotoxin action was accompanied by a 4.3-fold increase in the binding of [ 3H]batrachotoxinin A 20-α-benzoate to receptor site 2 on sodium channels in synaptosomes. These results point to an allosteric mechanism of brevetoxin action involving preferential binding to active states of sodium channels which have high affinity for neurotoxins, causing persistent activation of sodium channels at receptor site 2. Brevetoxin A does not block [ 3H]saxitoxin binding at neurotoxin receptor site 1 or 125I-labeled scorpion toxin binding at neurotoxin receptor site 3. The brevetoxins appear to act at a new neurotoxin receptor site on the sodium channel.

Potassium ion influx measurements on cultured Chinese hamster cells exposed to 60-hertz electromagnetic fields.

Potassium ion influx was measured by monitoring 42KCl uptake by Chinese hamster ovary (CHO) cells grown in suspension culture and exposed in the culture medium to 60-Hz electromagnetic fields up to 2.85 V/m. In the presence of the field CHO cells exhibited two components of uptake, the same as previously observed for those grown under normal conditions; both these components of influx were decreased when compared to sham-exposed cells. Although decreases were consistently observed in exposed cells when plotted as loge of uptake, the differences between the means of the calculated fluxes of exposed and sham-exposed cells were quite small (on the order of 4-7%). When standard deviations were calculated, there was no significant difference between these means; however, when time-paired uptake data were analyzed, the differences were found to be statistically significant. Cells exposed only to the magnetic field exhibited similar small decreases in influx rates when compared to sham-exposed cells, suggesting that the reduction in K+ uptake could be attributed to the magnetic field. Additionally, intracellular K+ levels were measured over a prolonged exposure period (96 h), and no apparent differences in intracellular K+ levels were observed between field-exposed and sham-exposed cultures. These results indicate that high-strength electric fields have a small effect on the rate of transport of potassium ions but no effect on long-term maintenance of intracellular K+.

Membrane permeability of secondary hydatid cysts of Echinococcus granulosus. Determination of the water diffusional and osmotic permeability coffficients thorugh a syncytial membrane

Diffusional ( P w) and osmotic ( P f) water permeability coefficients were determined for the syncytial epithelium of larval Echinococcus granulosus. P w was calculated from simultaneous influx measurements of tritiated water and n-[ 14 C] butanol through the hydatid cyst wall. The total diffusional water permeability coefficient, P′ w, was found to be 2.2 × 10 −4 cm s −1; which is similar to that previously reported by Rotunno et al. (1974, J. Parasitol. 60, 13–620). Nevertheless, when P′ f is corrected for the unstirred water layer effects, a P w value of 6.2 × 10 −4 cm s −1 is obtained. Thus, the unstirred water layer effects have a very important contribution to P′ w. Total steady state osmotic permeability coefficient, P′ f, was bound to be about 15 × 10 −4 cm s −1 and it is scarcely affected by those mechanisms that tend to distort the evaluation of P f. The experimentally determined osmotic coefficient differs from the corrected P f by only 6%. The P f/ P w ratio was found to be 2.4. The present study clearly confirms that syncytial membranes can be highly permeable to water, in spite of the fact that they lack tight junctions. Thus, water permeability through epithelial syncytium must be exclusively controlled by the permeability of the apical and/or basocellular membranes.

Numerical analysis of the method of internal dialysis of giant axons

This paper presents a numerical analysis of the method of internal dialysis used for studies of membrane transport in giant axons. Account is taken of the complete geometry, end effects, and finite dialyzate flow rates. Both influx and efflux experimental conditions are considered. Results place quantitative limits on system performance that are sufficiently general for use in experimental design. The completeness of solute equilibration and the uniformity of solute concentration at the axon membrane are assessed, as well as the sensitivity to dialysis solution flow rate. The effects of undialyzed axon ends on the equilibration rate and on the uniformity of concentration are determined, and the contribution of undialyzed ends to influx measurements is evaluated. Equilibration times are correlated with physical properties of axoplasm and dialysis tubing, and with dialysis solution flow rate. Numerical results confirm the general qualitative assessments of the method that are based on years of successful application.

Polyamines do not inhibit erythrocyte ATPase activities

To test whether physiologic elevation of red cell polyamine levels might explain Na pump inhibition in sickle cells or uremic red cells, we have studied the effect of putrescine, spermidine and spermine on red cell membrane ATPase and Na-K active transport. Measurement of the ouabain-sensitive influx of 86Rb into intact cells showed no effect of spermine. However, cells became depleted of ATP during incubation with spermine. By 48 h, the cells showed substantial potassium loss and moderate sodium gain. Because the low permeability of red cell membranes for polyamines might have obscured some direct effects on intracellular processes, we measured active transport of 22Na out of red cell ghosts that had been resealed in the presence of 5 mmol/1 spermine. In addition, we measured the Na-K, Mg, and Ca ATPase activities of broken membrane preparations in the presence of spermine, spermidine and putrescine. Polyamines had no direct effect on cation transport in red cells, although possible adverse effects on red cell metabolism could have a secondary effect on cation regulation.

Kinetics of tryptophan influx into brain slices depleted of sodium and loaded with L-histidine.

The kinetics of tryptophan influx were studied with rat brain slices preloaded with L-histidine and/or depleted of sodium ions. The best fits of the data (velocity of influx versus tryptophan concentration) were computed by use of a model consisting of a saturable (Michaelis-Menten type) and an unsaturable (diffusional) component with an iterative nonlinear regression analysis. Sodium depletion of the slices reduced the maximal velocity of saturable influx. In histidine-preloaded slices, depleted or not depleted of sodium ions, the most marked alteration again occurred in the maximal velocity, which more than doubled. Slices preloaded with histidine contained greatly elevated levels of glutamine and histidine, which may have stimulated the influx by exchange with extracellular tryptophan even in the absence of sodium ions. The maximal velocity was higher with increasing concentration of large neutral amino acids in slices at the start of the influx measurements. The influx of tryptophan in brain cells is apparently modified by changes in the intracellular amino acid pool, which, when increased, also counteracts the effect of sodium depletion on the tryptophan influx.

Two-compartment behavior during transport of folate compounds in L1210 cell plasma membrane vesicles.

The transport of [3H] 1,L 5-formyltetrahydrofolate, [3H] folic acid, and [3H]methotrexate by L1210 cell plasma membrane vesicles exhibited multicompartmental behavior. Two separate vesicular compartments (parallel relationship) of approximately equal volume were revealed during measurements of influx and efflux. Flux in one compartment was rapid, saturable, highly temperature-sensitive, and inhibited by pCMBS. Flux in the other compartment exhibited all of the characteristics of passive diffusion. These results imply that our plasma membrane vesicle preparations consist of a mixture of two functional species. Transport of folate into one of these species occurs by passive diffusion alone, whereas transport into the other kind of vesicle occurs by both passive diffusion and carrier-facilitated transport.

Effect of Triton X-100 on Tegument and Muscle in Schistosoma mansoni

Short periods of in vitro incubation of Schistosoma mansoni with low concentrations of the nonionic detergent Triton X-100 caused marked changes in the physiological and morphological status of the parasite. Less than 15 min of exposure to 1 mM Triton at 37 C led to irreversible destruction of the tegument, relaxation and depolarization of the muscle, and loss of response to electrical stimulation, to 60 mM K+-HBS, or to 10(-6) M praziquantel. A 5-min incubation in 100 microM Triton X-100 caused no gross structural damage, changes in tegumental or E2 electrical potential, or decrease in response to praziquantel, but markedly inhibited the ability of 60 mM K+-HBS or electrical stimulation to cause muscle contraction. Radioactive calcium influx measurements indicated that a 5-min exposure to 100 microM Triton X-100 may be uncoupling membrane depolarization from development of muscle tension by interfering pharmacologically with calcium channels. Longer incubations (15 min) in the presence of 100 microM Triton X-100 caused transitional physiological changes suggesting that the mode of action of the detergent was passing from a specific, pharmacological mode to a nonspecific disruptive action.

Unstirred Mucus Layers: Ion Exchange Properties and Effect on Ion Regulation in Lymnaea Stagnalis

ABSTRACT Mucus from the footsole of the freshwater snail Lymnaea stagnate behaves as a weak, negatively charged ion exchanger. Activities and concentrations of Na, K, Ca, and Cl were measured in mucus dialysed to equilibrium against artificial pond water or physiological saline. Observed activity coefficients (activity/concentration) in mucus were compared with those predicted by the Debye-Huckel theory to interpret the effects of electrostatic forces between the polyelectrolyte ions and small ions. The affinity of mucus for small ions decreased in the series, Ca2+, K+, Na+, Cl−. The extent to which mucus can concentrate cations was measured using three different methods: by titrating the fixed acidic groups with K or Ca and by equilibrium dialysis after which the electrical potential difference was either measured directly or was calculated from the Nernst potential for Na. Ion exchange titration indicated a much smaller exchange capacity than did the other two methods. Kinetics of cation uptake by the snail from dilute media were re-interpreted by considering the enhanced concentrations of cations in the mucus layer. It was shown that the presence of mucus in the unstirred layer adjacent to a transporting epithelium can result in an underestimate of the Michaelis constant (Km) determined from influx measurements.

K+ transport in "tight' epithelial monolayers of MDCK cells.

Bidirectional transepithelial K+ flux measurements across 'high-resistance' epithelial monolayers of MDCK cells grown upon millipore filters show no significant net K+ flux. Measurements of influx and efflux across the basal-lateral and apical cell membranes demonstrate that the apical membranes are effectively impermeable to K+. K+ influx across the basal-lateral cell membranes consists of an ouabain-sensitive component, an ouabain-insensitive component, an ouabain-insensitive but furosemide-sensitive component, and an ouabain- and furosemide-insensitive component. The action of furosemide upon K+ influx is independent of (Na+ - K+)-pump inhibition. The furosemide-sensitive component is markedly dependent upon the medium K+, Na+ and Cl- content. Acetate and nitrate are ineffective substitutes for Cl-, whereas Br- is partially effective. Partial Cl- replacement by NO3 gives a roughly linear increase in the furosemide-sensitive component. Na+ replacement by choline abolishes the furosemide-sensitive component, whereas Li+ is a partially effective replacement. Partial Na+ replacement by choline abolishes the furosemide-sensitive component, whereas Li+ is a partially effective replacement. Partial Na+ replacement with choline gives an apparent affinity of approximately 7 mM Na, whereas variation of the external K+ content gives an affinity of the furosemide-sensitive component of 1.0 mM. Furosemide inhibition is of high affinity (K1/2 = 3 micrometer). Piretanide, ethacrynic acid, and phloretin inhibit the same component of passive K+ influx as furosemide; amiloride, 4,-aminopyridine, and 2,4,6-triaminopyrimidine partially so. SITS was ineffective. Externally applied furosemide and Cl- replacement by NO3- inhibit K+ efflux across the basal-lateral membranes indicating that the furosemide-sensitive component consists primarily of K:K exchange.

Cellular distribution of neutral and basic amino acid transport systems in rabbit ileal mucosa.

1. An autoradiographic technique is described whereby the cellular location of tritiated amino acids can be determined following uptake by rabbit ileal mucosa. 2. Stirring solutions in contact with the intestinal mucosa during measurement of rapid influx changes the quantity, but not the distribution, of alanine taken up by the tissue. 3. Conditions predicted to favour either a high affinity system (Ly1) or a low affinity system (Ly2) were used to measure lysine distribution following uptake. Maximal uptake for both transport systems occurred in fully differentiated enterocytes at the tips of villi. Initial maturation of the Ly1 system, which was slow, was followed by a rapid phase of development. The Ly2 system lacked this rapid phase of late development. 4. The cellular distribution of alanine entering on a low affinity Na-independent neutral amino acid carrier closely resembles that determine for Ly1 system for lysine entry. 5. Arginine is a potent inhibitor of lysine uptake through the Ly2 system. Little or no diffusion of lysine appears to take place into rabbit ileal enterocytes. 6. The different distribution of the high and low affinity systems for lysine transport provides further support for their independent existence. It also suggests that more than one message exists fo the switching on of amino acid transport function in differentiating enterocytes.

Analysis of calcium handling in erythrocyte membranes of genetically hypertensive rats.

Calcium handling by erythrocyte membranes was compared in genetically hypertensive (SHR) and normotensive (WKR) rats by direct measurement of calcium binding, passive influx, and adenosine triphosphate (ATP)-dependent extrusion. The SHR erythrocyte membranes exhibited the following abnormalities: 1) the binding capacity of the high affinity Ca2+-binding sites located on the inner side of the membrane was 0.84 +/- 0.07 nmole/mg protein compared with 1.17 +/- 0.08 nmole/mg protein in WKR, 2) ATP-dependent Ca2+ extrusion, measured as the Ca2+ influx into inside-out vesicles, was also lower than the WKR, as was the La3+ -sensitive, Ca2+ -dependent hydrolysis, indicating reduced activity of the calcium pump; 3) the passive calcium influx into ATP-depleted red blood cells was slightly accelerated. these abnormalities in Ca2+ binding and transport probably enhanced intracellular Ca2+ concentration, and were observed under both prehypertensive an hypertensive conditions, in 3-week-old and adult SHR respectively. Similar membrane defects in excitable cells may help to explain the pathogenesis of hypertension, since they may increase vascular tone and/or catecholamine release.

Growth and uptake rates of P, K, Ca and Mg in wheat1

Abstract The relation between plant age and nutrient absorption properties of red winter wheat (Triticum aestivum L.) roots were investigated. Understanding the change in ion uptake parameters with increasing plant age is helpful in devising efficient fertilization systems. Such information can be used to determine the nutrient levels needed in the soil to supply nutrients rapidly enough to the root surface to minimize deficiencies. Wheat was grown for periods up to 40 days in solution culture in a controlled climate chamber. Sequential harvest and nutrient influx measurements were made. Shoot growth was exponential with time to 30 days and linear thereafter. Root dry weight increased linearly with time at a slower rate than shoot dry weight. Root length increased linearily with time. With increasing plant age there was a reduction in average P and K uptake rate while average uptake rates for Ca and Mg remained relatively unchanged. With increasing plant age, the maximum influx, Imax. for P and Mg remaine...

Influence of extracellular Cl concentration on Cl transport across isolated skin of Rana pipiens.

The effect of changes in Cl concentration in the external and/or serosal bath on Cl transport across short-circuited frog skin was studied by measurements of transepithelial Cl influx (JCl13) and efflux (JCl31), short-circuit current, transepithelial potential, and conductance (Gm). JCl13 as well as JCl31 were found to have a saturating component and a component which is apparently linear with Cl concentration. The linear component of JCl31 appears only upon addition of Cl to external medium, and about 3/4 of this component does not contribute to Gm. The saturating component of JCl31 is only 5% of total JCl31 with 115 mM Cl in the serosal medium. Replacement of 115 mM Cl- in external medium by SO4=, NO3-, HCO-3 or I- results in 87-97% reduction of JCl31, whereas replacement with Br- has no effect. As external Cl concentration is raised in steps from 2 to 115 mM, JCl13 and JCl31 increase by the same amount but JCl13 is persistently 0.15 mu eq/cm2hr larger than JCl31. These results indicate that at least 3/4 of linear components of JCl13 and JCl31 proceed via an exchange diffusion mechanism which seems to be located at the outer cell border. The saturating component of JCl13 is involved in active Cl transport in an inward direction, and there is evidence suggesting that Cl uptake across outer cell border, which proceeds against an electrochemical gradient, is electroneutral but not directly linked to Na.

Effects of dietary intake of sodium chloride on sugar and amino acid transport across isolated hen colon.

1. Using the isolated mucosa from the colon of the adult hen, transport of galactose, leucine and lysine was studied through measurements of influx across the brush-border membrane, unidirectional transmucosal fluxes, and of steady-state mucosal uptake.2. In hens maintained on a NaCl rich diet influx of galactose, leucine and lysine were saturable processes with well defined values for J(max) and K(t). All three substances were actively transported across the epithelium and accumulated in the mucosal tissues to steady-state concentrations several times higher than in the incubation media.3. In hens maintained on a NaCl poor diet the K(t) of the leucine influx was markedly increased and the J(max) moderately decreased, while the epithelium maintained the capability of active transepithelial transport and of establishing a steady-state tissue/medium distribution ratio well above 1. Galactose and lysine could no longer be actively transported and in the steady state their tissue concentrations did not exceed those of the medium.4. It is proposed that in the low NaCl colon the processes of galactose and lysine co-transport with Na are abolished and that the transport of galactose proceeds via a phloretin sensitive system like that described for the enterocyte of the chicken small intestine.5. In the high NaCl colon 1 mM-leucine increases the mucosa to serosa flux of lysine by a factor 2.8 without affecting the serosa to mucosa flux or the steady-state mucosal accumulation of lysine. In the low NaCl colon 1 mM-leucine induces active transepithelial transport of lysine. Assuming that the active transport of leucine is secondary to the transport of Na, leucine-induced active transport of lysine is described as a tertiary active transport.6. In the high NaCl colon the mucosa to serosa flux and the steady-state mucosal accumulation of leucine were inhibited by galactose which had no effect on leucine influx across the brush-border membrane. Leucine did not affect any of the parameters of galactose transport.