Mechanisms of chloride influx during KCl-induced swelling in the chicken retina

An increase in extracellular KCl ([KCl]_o) occurs under various pathological conditions in the retina, leading to retinal swelling and possible neuronal damage. The mechanisms of this KCl_o-induced retinal swelling were investigated in the present study, with emphasis on the Cl^– transport mechanisms. Increasing [KCl]_o (from 5 to 70 mM) led to concentration-dependent swelling in chicken retinas. The curve relating the degree of swelling to [KCl]_o was biphasic, with one component from 5 to 35 mM [KCl]_o and another from 35 to 100 mM. As Cl^– omission prevented swelling in all conditions, the effect of cotransporter or Cl^– channel blockers was examined to investigate the mechanisms of Cl^– influx. The cotransporter blockers bumetanide and DIOA reduced swelling by 68% and 76%, respectively at [KCl]_o 25 mM (K25), and by 14–17% at [KCl]_o 54 mM (K54). The Cl^– channel blockers NPPB and niflumic acid did not affect swelling at K25 but reduced it by 90–95% at K54 (NPPB IC₅₀ 60.7 µM). Furosemide showed an atypical effect, decreasing swelling by 14% at K25 and by 95% at K54 (IC₅₀ 173.9 µM). Na⁺ omission decreased swelling at K25 but not at K54. These results suggest the contribution of cotransporters to Cl^– influx at K25 and of Cl^– channels at K54. At K25, swelling was found in the ganglion cell layer and in the lower half of the inner nuclear layer. With K54, swelling occurred in all inner retinal layers. The ganglion cell layer swelling was due to both Müller cell end-foot and ganglion cell soma swelling. K54 also induced ganglion cell damage as shown by disorganized, pyknotic and refringent nuclei.     

Erythrocyte sodium content and transport in borderline and mild hypertension

Summary Sodium content and transport of red blood cells were examined in 98 male blood donors. Regarding their blood pressure they were classified into the following groups: (a) 57 normotensives, (RR<140/90 mm Hg); (b) 24 borderline hypertensives (140/90RR<160/95 mm Hg); and (c) 17 hypertensives (RR>160/100 mm Hg). Compared with the normotensives the borderline hypertensives have significantly reduced red cell sodium content. The ouabain-resistant net Na⁺ uptake and the relative Na⁺ uptake, as a measure of the Na⁺/K⁺ pump, were significantly increased. With rising blood pressures the measured values turn to normal, so that no difference exists between the normotensive and hypertensive groups. It is supposed that in the initial or even prehypertensive state a considerable enhancement of the pump activity occurs, simultaneously accompanied by less marked increases in sodium influx, leading to a reduced intracellular sodium content. In the course of hypertension, possibly caused by the formation of a pump inhibitor, the sodium content of red cells turns to normal or supernormal values.Abbreviations BMI body mass index - BHT borderline hypertensive - Ca _ion ²⁺ ionized plasma calcium - HT hypertensive - k relative OR net Na⁺ uptake - [Na⁺]_i, [K⁺]_i intracellular sodium and potassium content in RBCs - NT normotensive - OR ouabain-resistant - RBCs red blood cells - Na OR net Na⁺ uptake     

Relation of exocytotic release of γ-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the -aminobutyric acid (GABA) carrier, NNC-711, {1-[(2-diphenylmethylene) amino]oxyethyl}-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride, blocks the Ca²⁺-independent release of [³H]GABA from rat brain synaptosomes induced by 50 mM K⁺ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca²⁺-dependent release of [³H]GABA in the total absence of carrier-mediated release. Reversal of the Na⁺/Ca²⁺ exchanger was used to increase the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) to test whether an increase in [Ca²⁺]_i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca²⁺]_i may rise to values above 400 nM, as a result of Na⁺/Ca²⁺ exchange, without inducing release of [³H]GABA, but subsequent K⁺ depolarization immediately induced [³H]GABA release. Thus, a rise of only a few nanomolar Ca²⁺ in the cytoplasm induced by 50 mM K⁺ depolarization, after loading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange, induced exocytotic [³H]GABA release, whereas the rise in cytoplasmic [Ca²⁺] caused by reversal of the Na⁺/Ca²⁺ exchanger was insufficient to induce exocytosis, although the value for [Ca²⁺]_i attained was higher than that required for exocytosis induced by K⁺ depolarization. The voltage-dependent Ca²⁺ entry due to K⁺ depolarization, after maximal Ca²⁺ loading of the synaptosomes by Na⁺/Ca²⁺ exchange, and the consequent [³H]GABA release could be blocked by 50 M verapamil. Although preloading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange did not cause [³H]GABA release under any conditions studied, the rise in cytoplasmic [Ca²⁺] due to Na⁺/Ca²⁺ exchange increased the sensitivity to external Ca²⁺ of the exocytotic release of [³H]GABA induced by subsequent K⁺ depolarization. Thus, our results show that the vesicular release of [³H]GABA is rather insensitive to bulk cytoplasmic [Ca²⁺] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca²⁺ entry through Ca²⁺ channels near the active zones.     

Determination of extracellular space and intracellular electrolytes in rat liver in vivo

1. Uptake of a number of isotopically labelled extracellular tracers by rat liver in vivo was studied.

2. A residual intravascular space of 3·0-3·5% following exsanguination was measured by use of [⁵¹Cr]red cells. This space was significantly greater than that of skeletal muscle and was also greater in nephrectomized as compared to intact rats.

3. [¹³¹I]albumin and polyvinylpyrolidine (PVP) were found to distribute initially in a space of 6-7% and this was followed by a delayed rise that was greater for PVP. The extravascular portion of this space is termed the large-molecule accessible space and is believed to correspond to the histological space of Disse.

4. [¹⁴C]sucrose and inulin initially distribute in a space of 10-12% followed by a continued late increase. This initial space is termed the small molecule accessible space and is thought to include most of interstitial space.

5. ³⁶Cl^- and stable Na⁺ and Cl^- spaces were constant at 19-21% and can be used to put an upper limit on extracellular space.

6. Operationally, the 1 hr inulin space in the nephrectomized rat was felt to be the best indicator of extracellular space.

7. Total electrolytes were measured and intracellular values of 22, 165 and 22 m-mole/l. cell water for Na⁺, K⁺ and Cl^-, respectively, were calculated.

8. By use of [¹⁴C]DMO, an intracellular pH of 7·25 and an intracellular HCO₃^- of 13 m-mole/l. cell water were calculated.

     

Spatial and temporal control of intracellular free Ca2+ in chick sensory neurons

Digital imaging of fura-2 fluorescence and the voltage-clamp technique were combined to study cytoplasmic free Ca²⁺ concentration, [Ca]_i, in neurons cultured from chick dorsal root ganglia. Depolarizing pulses raised [Ca]_i to a new steady-state level which was achieved earlier in neurites than in the soma. The rise in [Ca]_i during stimulated bursting or rhythmic activity was also faster in neurites. After stimulation [Ca]_i recovered monoexponentially in the soma and biexponentially in neurites. Application of 50 mM KCl produced membrane depolarization and a concomitant increase of [Ca]_i. During wash-out [Ca]_i often declined to an intermediate steady-state level at which it stayed for several minutes. Thereafter the resting level of [Ca]_i was quickly restored. [Ca]_i recovery was delayed after treating the cell with 2 M thapsigargin, an inhibitor of the Ca²⁺ pump of internal Ca²⁺ stores. Caffeine (10 mM) transiently increased [Ca]_i. A second caffeine application produced smaller [Ca]_i changes due to the prior depletion of Ca²⁺ stores, which could be replenished by brief exposure to KCl. Thapsigargin (2 M) transiently increased [Ca]_i both in the standard and Ca²⁺-free solution. [Ca]_i transients due to caffeine and thapsigargin started in the cell interior, in contrast to [Ca]_i changes evoked by membrane depolarization, which were noticed first at the cell edge. Caffeine and thapsigargin induced a transient inward current which persisted in the presence of 1 mM La³⁺ and in Ca²⁺-free solutions, but which was greatly diminished in Na⁺-free solutions. The effects of caffeine and thapsigargin were mutually exclusive both in the generation of [Ca]_i transients and in the inward current induction.     

Modest intracellular acidification suppresses death signaling in ouabain-treated cells

The signaling cascade resulting in the death of several types of cells treated with ouabain or other cardiotonic steroids (CTS) remains poorly understood. Recently, we observed that ouabain kills epithelial and endothelial cells via its interaction with Na⁺, K⁺ -ATPase, but independently of inhibition of Na⁺, K⁺ -ATPase-mediated ion fluxes and inversion of the [Na⁺]_i/[K⁺]_i ratio. Here, we report that the death of ouabain-treated epithelial cells from the Madin-Darby canine kidney (C7-MDCK) and endothelial cells from porcine aortae is suppressed by acidification of medium from pH 7.4 to 7.0, i.e. under conditions when pH_i was decreased from 7.2 to 6.9. The rescue of ouabain-treated C7-MDCK cells was also detected under selective intracellular acidification caused by inhibition of Na⁺/H⁺ exchanger. In these cells, neither Na⁺, K⁺ pump activity nor [³H]-ouabain binding was significantly affected by modest acidification. The death of ouabain-treated cells was independent of inhibition of RNA and protein synthesis with actinomycin D and cycloheximide. In contrast, both compounds sharply attenuated the protective action of acidified medium. Thus, our results show that very modest intracellular acidification is sufficient to inhibit the Na⁺ _i/K⁺ _i-independent death signal triggered in epithelial and endothelial cells by CTS. They also suggest that the protective action of acidification is mediated by de novo expression of genes involved in inhibition of the cell death machinery.     

Intracellular Ca2+ transients in HT29 cells induced by hypotonic cell swelling

Cell swelling induced by hypotonic solution led to an osmolality-dependent increase in intracellular Ca²⁺ activity ([Ca²⁺]_i) in HT₂₉ cells. At moderate reductions in osmolality from 290 to 240 or 225 mosmol/l in most cases only a small monophasic increase of [Ca²⁺]_i to a stable plateau of 10–20 nmol/l above resting [Ca²⁺]_i was observed. Lower osmolalities resulted in a triphasic increase of [Ca²⁺]_i to a peak value. In a first phase after the volume change, lasting 20–40 s, [Ca²⁺]_i increased slowly by about 30 nmol/l. Thereafter [Ca²⁺]_i increased more rapidly within 20–30 s to a peak value. This peak was 189±45 nmol/l (190 mosmol/l, n=9) and 243±41 nmol/l (160 mosmol/l, n=20) above resting [Ca²⁺]_i. The peak was then followed by a decline of [Ca²⁺]_i over the next 2–3 min to a stable plateau value of 28±6 (n=6) and 32±11 nmol/l (n=11) above resting [Ca²⁺]_i at 190 and 160 mosmol/l, respectively. The plateau lasted as long as the hypotonic solution was present. Under Ca²⁺-free bath conditions the peak value for the cell-swelling-induced [Ca²⁺]_i transient was reached significantly later (60–100 s, compared to 40–60 s under control conditions). The peak values under Ca²⁺-free conditions were not significantly lower. This indicates that the [Ca²⁺]_i peak was mostly of intracellular origin. No [Ca²⁺]_i plateau phase was observed under Ca²⁺-free bath conditions. With the use of the fura-2-Mn ²⁺ quenching technique an increased Ca²⁺ influx induced by hypotonic cell swelling was shown (160 mosmol/l; n=4). This influx started immediately after or simultaneously with the cell swelling and preceded the [Ca²⁺]_i peak for more than 50 s.This study was supported by DFG grant Gr 480/10.     

An efficient and simple method of non-enzymatic detachment of monolayer cultures into single cell suspension

Summary Activating the Na⁺/H⁺ antiporter causes a distinctive and rapid endocytosis which internalizes the plasma membrane leading to cell retraction and detachment as rounded cells with much reduced surface area. Antiport activation is by a combination of two individually effective motivations, viz. a) steep [Na⁺] and [H⁺] gradients across the plasma membrane and b) allosteric activation by second messengers initiated with simple inorganic sulphate. The dissociated cells are as viable as those released by trypsinization. Thus it provides an effective enzyme-free alternative to trypsin digestion in cell dissociation.     

The intracellular Ca2+ concentration optimal for T cell activation is quite different after ionomycin or CD3 stimulation

The relationship between the initial increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) (measured at the single-cell level with an imaging system) and the ensuing proliferation was examined in a human T cell clone stimulated by a phorbol ester in combination with ionomycin, thapsigargin or an anti-CD3 mAb (monoclonal antibody against the CD3 molecule, UCHT1). From the responses to various ionomycin concentrations, one can define a range of [Ca²⁺]_i values (400–900 nM) which appears optimal for T cell proliferation; lower [Ca²⁺]_i values are suboptimal, higher values are cytotoxic. It was then examined if the [Ca²⁺]_i requirements were similar following anti-CD3 stimulation. [Ca²⁺]_i oscillations elicited by a concentration of UCHT1 (1/1,000) optimal for mitogenicity fall precisely within the 400–900 nM range. However, very low concentrations of UCHT1 (1/100,000) which evoke barely detectable [Ca²⁺]_i responses still cause the cells to proliferate. The possibility that the lower [Ca²⁺]_i requirements observed following anti-CD3 stimulation was due to [Ca²⁺]_i oscillations was tested under conditions which prevented the appearance of these oscillations. It turns out that an oscillatory Ca²⁺signal is not more mitogenic than a sustained augmentation of [Ca²⁺]_i. Finally, it was examined if overstimulation via CD3 could have toxic consequences similar to those elicited after ionomycin overstimulation. Large transient [Ca²⁺]_i responses can be observed following anti-CD3 stimulation in appropriate conditions, and namely in T cells pretreated with interleukin-2. These [Ca²⁺]_i augmentations are not cytotoxic. A role for the plasmalemmal Ca²⁺ pump in the prevention of cytotoxicity can be demonstrated. In conclusion, the correspondence between the [Ca²⁺]_i response and cell proliferation is entirely different following stimulation by ionomycin and by anti-CD3. In addition, cell proliferation evoked by very low UCHT1 concentration might reveal the existence of a yet unidentified activation pathway.     

In vivo evaluation of amyloid deposition and brain glucose metabolism of 5XFAD mice using positron emission tomography

Positron emission tomography (PET) has been used extensively to evaluate the neuropathology of Alzheimer's disease (AD) in vivo. Radiotracers directed toward the amyloid deposition such as [¹⁸F]-FDDNP (2-(1-{6-[(2-[F]Fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile) and [¹¹C]-PIB (Pittsburg compound B) have shown exceptional value in animal models and AD patients. Previously, the glucose analogue [¹⁸F]-FDG (2-[(18)F]fluorodeoxyglucose) allowed researchers and clinicians to evaluate the brain glucose consumption and proved its utility for the early diagnosis and the monitoring of the progression of AD. Animal models of AD are based on the transgenic expression of different human mutant genes linked to familial AD. The novel transgenic 5XFAD mouse containing 5 mutated genes in its genome has been proposed as an AD model with rapid and massive cerebral amyloid deposition. PET studies performed with animal-dedicated scanners indicate that PET with amyloid-targeted radiotracers can detect the pathological amyloid deposition in transgenic mice and rats. However, in other studies no differences were found between transgenic mice and their wild type littermates. We sought to investigate in 5XFAD mice if the radiotracers [¹¹C]-PIB, and [¹⁸F]-Florbetapir could quantify the amyloid deposition in vivo and if [¹⁸F]-FDG could do so with regard to glucose consumption. We found that 5XFAD animals presented higher cerebral binding of [¹⁸F]-Florbetapir, [¹¹C]-PIB, and [¹⁸F]-FDG. These results support the use of amyloid PET radiotracers for the evaluation of AD animal models. Probably, the increased uptake observed with [¹⁸F]-FDG is a consequence of glial activation that occurs in 5XFAD mice.     

Changes in Na+, K+-adenosinetriphosphatase,citrate synthase and K+ in sheep skeletal muscle during immobilization and remobilization

The K⁺ balance and muscle activity seem to interact in a complex way with regard to regulating the muscle density of Na⁺-K⁺ pumps. The effect of immobilization was examined in ten sheep that had low muscle K⁺ content. Three additional sheep served as untreated controls. After being brought from pasture to sheep stalls one hindlimb was immobilized in a plaster splint for 9 weeks, and in five of the animals remobilization was carried out for a further 9 weeks. The weight bearing of the leg in plaster was recorded by a force plate. Open muscle biopsies from the vastus lateralis muscle were obtained before the study, after 9 weeks of immobilization, and after another 9 weeks of remobilization. The Na⁺-K⁺ pump density was measured as [³H]-ouabain binding to intact tissue, and citrate synthase activity was measured in tissue homogenate. The tissue content of K⁺ was measured in fat-free dried tissue. Muscle K⁺ content increased linearly by almost 70% through the 18-week period independent of intervention. Immobilization reduced thigh circumference by 8% (P < 0.05) . A slight decrease in the area of type I fibres at 9 weeks and a slight increase at 18-weeks was found. The [³H]-ouabain binding was reduced by 39% and 22% in the immobilized and control legs, respectively, whereas citrate synthase activity was reduced by about 30% in both legs after 9 weeks of immobilization. During remobilization both the [³H]-ouabain binding and the citrate synthase activity increased to the same level as in the control animals. The plaster cast significantly reduced mass bearing of the immobilized leg, and a corresponding reduction in muscle activity must be assumed to have occurred in both legs as judged from citrate synthase activity. We concluded from this study that the reduction in the [³H]-ouabain binding during immobilization independent of an increase in muscle K⁺ content points to muscle activity as a strong stimulus for control of Na⁺-K⁺ bump density.     

Functional characterization of the Ca2+-gated Ca2+ release channel of vascular smooth muscle sarcoplasmic reticulum

The Ca²⁺-gated Ca²⁺ release channel of aortic sarcoplasmic reticulum (SR) was partially purified and reconstituted into planar lipid bilayers. Canine and porcine aorta microsomal protein fractions were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) in the presence and absence of ³[H]-ryanodine and centrifuged through linear sucrose gradients. A single ³[H]-ryanodine receptor peak with an apparent sedimentation coefficient of 30 s was obtained. Upon reconstitution into planar lipid bilayers, the unlabelled 30 s protein fraction induced the formation of a Ca²⁺- and monovalent-ion-conducting channel (110 pS in 100 mM Ca²⁺, 360 pS in 250 mM K⁺). The channel was activated by micromolar Ca²⁺, modulated by millimolar adenosine triphosphate, Mg²⁺ and the Ca²⁺-releasing drug caffeine, and inhibited by micromolar ruthenium red. Micro- to millimolar concentrations of the plant alkaloid ryanodine induced a permanently closed state of the channel. Our results suggest that smooth muscle SR contains a Ca²⁺-gated Ca²⁺ release pathway, with properties similar to those observed for the skeletal and cardiac ryanodine receptor/Ca²⁺ release channel complexes.     

Erythrocyte cations and Na+,K+-ATPase pump activity in athletes and sedentary subjects

Summary The chronic effect of training on intraerythrocyte cationic concentrations and on red cell Na⁺,K⁺-ATPase pump activity was studied by comparing well-trained athletes with sedentary subjects at rest. Also the acute effect of a 50-min cross-country run on these erythrocyte measurements was studied in the athletes. At rest the intraerythrocyte potassium concentration was increased (P<0.01) in the athletes compared to that of the control subjects. The intraerythrocyte concentrations of sodium and magnesium and red cell Na⁺, K⁺-ATPase pump activity were, however, similar in the trained and the untrained subjects.As compared with the resting condition, the intraerythrocyte potassium concentration was decreased (P<0.05) after exercise in the athletes, and this was accompanied by a minor increase in the intraerythrocyte sodium concentration. Red cell Na⁺,K⁺-ATPase pump activity was slightly increased (P<0.05) after exercise.     

Interspersed normoxia during live high, train low interventions reverses an early reduction in muscle Na+, K+ATPase activity in well-trained athletes

Hypoxia and exercise each modulate muscle Na⁺, K⁺ATPase activity. We investigated the effects on muscle Na⁺, K⁺ATPase activity of only 5 nights of live high, train low hypoxia (LHTL), 20 nights consecutive (LHTLc) versus intermittent LHTL (LHTLi), and acute sprint exercise. Thirty-three athletes were assigned to control (CON, n = 11), 20-nights LHTLc (n = 12) or 20-nights LHTLi (4 × 5-nights LHTL interspersed with 2-nights CON, n = 10) groups. LHTLc and LHTLi slept at a simulated altitude of 2,650 m (F_IO₂ 0.1627) and lived and trained by day under normoxic conditions; CON lived, trained, and slept in normoxia. A quadriceps muscle biopsy was taken at rest and immediately after standardised sprint exercise, before (Pre) and after 5-nights (d5) and 20-nights (Post) LHTL interventions and analysed for Na⁺, K⁺ATPase maximal activity (3-O-MFPase) and content ([³H]-ouabain binding). After only 5-nights LHTLc, muscle 3-O-MFPase activity declined by 2% (P < 0.05). In LHTLc, 3-O-MFPase activity remained below Pre after 20 nights. In contrast, in LHTLi, this small initial decrease was reversed after 20 nights, with restoration of 3-O-MFPase activity to Pre-intervention levels. Plasma [K⁺] was unaltered by any LHTL. After acute sprint exercise 3-O-MFPase activity was reduced (12.9 ± 4.0%, P < 0.05), but [³H]-ouabain binding was unchanged. In conclusion, maximal Na⁺, K⁺ATPase activity declined after only 5-nights LHTL, but the inclusion of additional interspersed normoxic nights reversed this effect, despite athletes receiving the same amount of hypoxic exposure. There were no effects of consecutive or intermittent nightly LHTL on the acute decrease in Na⁺, K⁺ATPase activity with sprint exercise effects or on plasma [K⁺] during exercise.     

Epidermal growth factor stimulates Ca2+ uptake of human erythrocytes

To examine the functional significance of epidermal growth factor (EGF) binding sites present on the human erythrocyte membrane [Engelmann et al. (1992) Am J Hematol 39:239–241], the effect of EGF on ⁴⁵Ca²⁺ uptake and on ²²Na⁺ efflux from these cells has been studied. In all cases media contained 1.25 mM Ca²⁺, whereas Na⁺ and K⁺ were varied. In 140 mM Na⁺/5 mM K⁺ medium EGF (250 ng/ml) stimulated ⁴⁵Ca²⁺ uptake by 50%–90% in quin-2-loaded cells, and by up to threefold in untreated cells. Increasing extracellular K⁺ up to 75 mM at the expense of extracellular Na²⁺ stimulated the EGF-induced ⁴⁵Ca²⁺ uptake by about twofold compared to 145 mM Na⁺ medium both in quin-2-loaded and in untreated cells. In 145 mM K⁺ medium, however, no EGF-induced ⁴⁵Ca²⁺ uptake was detectable in quin-2-loaded cells, while in untreated cells Ca²⁺ entry was stimulated twofold by EGF. After increasing intracellular Na⁺ from 6 mmol/l cells to 18 mmol/l cells in untreated cells suspended in 145 mM K⁺ medium, ⁴⁵Ca²⁺ uptake induced by EGF gradually increased. In contrast, in 140 mM Na⁺/5 mM K⁺ as well as in 70 mM Na⁺/75 mM K⁺ medium, ⁴⁵Ca²⁺ uptake accelerated by EGF was largely unaffected by a modified red cell Na⁺ content. When ²²Na-loaded untreated red cells were suspended in 145 mM K⁺ medium EGF stimulated red cell ²²Na⁺ efflux by more than threefold. In 140 mM Na⁺/5 mM K⁺ as well as in 70 mM Na⁺/75 mM K⁺ medium, no ²²Na⁺ efflux induced by the growth factor was evident. The results are consistent with the idea that EGF stimulates (at least) two components of ⁴⁵Ca²⁺ uptake in human erythrocytes. One of the two is unmasked in 145 mM K⁺ medium, inhibited by quin-2 loading, accelerated by intracellular Na⁺ and appears to involve reversed Na⁺/Ca²⁺ exchange.     

Proportions of total blood iodide in the red cells of the rabbit

Summary 1. 321 measurements on the ¹³¹I-iodide content of the erythrocytes were carried out in 27 rabbits under different conditions. 21.48 (±2.49) per cent of the intravascular activity was found to be in the red cells of normal rabbits at a mean haematocrit value of 34.5%. Since the blood volume represents 18 per cent of the ¹³¹I-iodide space in the rabbit, 4.5% of this space can be regarded as part of the intravascular cellular compartment.2. No significant differences in the intracellular ¹³¹-I-iodide content were found in rabbits on excess iodide and chloride intake and also not in narcosis or after thyroidectomy or plasmapheresis, but there was a marked reduction of radioiodide in the red cells when the rabbit was previously subjected to repeated haemorrhages.3. The intravascular ¹³¹I-iodide distribution shows only a partial correlation with haematocrit values, the coefficient of this correlation being +0.802.4. If the distribution of radioiodide between plasma and erythrocytes is calculated on the basis of the plasma/red cell water ratio, in most cases relatively more ¹³¹I-iodide will be found in the plasma than in the erythrocytes. Plasma/red cell ¹³¹I-iodide ratios obtained in normal rabbits averaged 1.18 (±0.095).5. The results are statistically evaluated and briefly commented on.With 2 Figures in the Text     

Low [NaCl]-induced neuronal nitric oxide synthase (nNOS) expression and NO generation are regulated by intracellular pH in a mouse macula densa cell line (NE-MD)

Changes in the luminal NaCl concentration ([NaCl]) at the macula densa (MD) modulate the tubuloglomerular feedback (TGF) responses via an affect on the release of nitric oxide (NO). This study was performed in a newly established mouse macula densa cell line (NE-MD) to investigate the effects of lowering [NaCl] on the neuronal NO synthase (nNOS) protein expression and l-arginine (Arg)-induced NO release. Expression of nNOS protein and release of NO were evaluated by Western blot analysis and an NO-sensitive electrode, respectively. Intracellular pH (pH_i) was monitored by the BCECF assay. Although there was weak staining of the nNOS protein expression, l-Arg-induced NO generation was negligible in normal (140 mM NaCl) solution. Both were significantly (P < 0.05) increased either in the presence of furosemide (12 μM), an inhibitor of the Na⁺–K⁺–2Cl⁻ cotransporter, or in a low (23 mM) Cl⁻ solution. Furosemide- and low Cl⁻-induced NO generation was completely inhibited by 50 μM 7-nitroindasole (7-NI), a nNOS inhibitor. Moreover, these increases were significantly (P < 0.05) inhibited by the addition of 100 μM amiloride, an inhibitor of the Na⁺/H⁺ exchanger, or by its analogue 5-(N)-ethyl-N-isopropyl amiloride (EIPA), and also at a lower pH of 7.1. Furthermore, nNOS expression and NO release were not stimulated in as low as 19 mM Na⁺ solution. In conclusion, low [Cl⁻], but not low [Na⁺] in the lumen at the MD, increased nNOS protein expression and NO generation. Changes in the luminal [NaCl] may modulate the TGF system via an effect on the NO generation from the MD.     

Effect of olsalazine and mesalazine on human ileal and colonic (Na+ + K+)-ATPase

Summary Olsalazine (azodisalicylate) and mesalazine (5-aminosalicylic acid) have recently been developed as new treatment modalities for inflammatory bowel disease to avoid sulfasalazine-related side effects. However, there are reports regarding new and hitherto unexpected side effects in some patients receiving olsalazine or mesalazine, such as watery diarrhea. Since sodium pump activities play an important role in the pathogenesis of water and electrolyte disturbances, we investigated the influence of olsalazine and mesalazine on human ileal and colonic (Na⁺ + K⁺)-ATPase and its specific [³H]-ouabain binding. We found a concentration-dependent inhibition of ileal and colonic (Na⁺ + K⁺)-ATPase by olsalazine with an IC₅₀ of 4.1 mM and 4.8 mM, respectively. Mesalazine inhibited this enzyme in the ileum with an IC₅₀ of 4.5 mM and in the sigmoid colon with an IC₅₀ 3.5 mM. In addition, [³H]-ouabain binding was inhibited by mesalazine with an IC₅₀ of 3.6 mM. The maximal inhibition, however, did not exceed 80% under any conditions (up to 10 mM drug concentration). Olsalazine and mesalazine induce inhibition of the ileal and colonic sodium pump activities that may (in addition to other possible mechanisms) mediate impaired water and electrolyte absorption. This is possibly of clinical relevance in patients with severely damaged mucosa. In patients with milder forms of mucosal inflammation, this inhibition most likely is of minor importance because of the great capacitiy of the (Na⁺ + K⁺)-ATPase and the incomplete inhibition leaving at least 20% of the enzyme activity intact.Abbreviations 5-ASA 5-aminosalicylic acid - EDTA ethylenediaminetetracetic acid - IBD inflammatory bowel disease     

Na+/K+ pump and endothelial cell survival: [Na+]i/[K+]i-independent necrosis triggered by ouabain, and protection against apoptosis mediated by elevation of [Na+]i

Recent studies have demonstrated the tissue-specific effect of Na⁺/K⁺ pump inhibition by ouabain and other cardiac glycosides on cell viability. The vascular endothelium is an initial target of cardiac glycosides employed for the management of congestive heart failure as well as circulating endogenous ouabain-like substances (EOLS), the production of which is augmented in volume-expanded hypertension. This study examined the role of the Na⁺/K⁺ pump in the survival of cultured porcine aortic endothelial cells (PAEC). Complete Na⁺/K⁺ pump inhibition with ouabain led to PAEC death, indicated by cell detachment and decreased staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Based on cell swelling and resistance to benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk) a pan-caspase inhibitor, this type of cell death was classified as necrosis. In contrast to ouabain, Na⁺/K⁺ pump inhibition in K⁺-free medium did not affect PAEC viability and sharply attenuated apoptosis triggered by ³H decay-induced DNA damage. Necrosis evoked by ouabain was preserved after dissipation of the transmembrane gradient of K⁺ and Na⁺, whereas dissipation of the Na⁺ gradient abolished the antiapoptotic action of K⁺-free medium. Comparative analysis of these results and modulation of intracellular Na⁺ and K⁺ content by the above-listed stimuli showed that interaction of ouabain with Na⁺/K⁺-ATPase triggered necrosis independently of inhibition of Na⁺/K⁺ pump-mediated ion fluxes and inversion of the [Na⁺]_i/[K⁺]_i ratio, whereas protection against apoptosis under Na⁺/K⁺ pump inhibition in K⁺-depleted medium was mediated by [Na⁺]_i elevation. The role of Na⁺/K⁺ pump-mediated regulation of endothelial cell survival and vascular remodelling seen in hypertension should be investigated further in context of EOLS and chronic treatment with digitalis.     

Relationship between cell volume and cation content in thick ascending limb of rat kidney

We examined the relationship between the cell volume and cation concentration ([Na_i] and [K_i]) of isolated segments of rat medullary thick ascending limb (MAL) after incubation at 30°C in various isotonic solutions. When the tubules were incubated in a normal NaCl solution containing 5 mmol/l K⁺, addition of 1 mmol/l of ouabain increased [Na_i] and decreased [K_i] but did not change the total ([Na_i]+[K_i]) concentration (about 90 mEq/l) or tubular volume. After incubation in various K⁺-free solutions, the tubules were almost fully K⁺-depleted; their volume per unit of length was similar in the three solutions, although the choline Cl-treated tubules had a very low sodium content compared to the NaCl-and Na₂SO₄-treated tubules (8 vs. 97 and 95 mEq/l respectively). Ouabain altered neither volume nor [Na_i] of tubules incubated in choline Cl or Na₂SO₄ solution. Transfer of tubules from K⁺-free Na₂SO₄ or K⁺-free choline Cl solution into K⁺-free NaCl solution resulted in an increase in [Na_i] (by 29 and 97 mEq/l respectively) without much increase in tubular volume. A marked swelling of the tubules was only observed when the K⁺-free NaCl solution contained also ouabain. Under this condition, [Na_i] was comparable to the Na⁺ concentration of the incubation medium. After washing and incubation in a normal NaCl solution containing K⁺, the swollen tubules recovered their initial volume and restored Na⁺ and K⁺ concentration gradients across the cell membranes. The ([Na_i]+[K_i]) concentration centration measured in the tubules preincubated in choline Cl solution was always smaller than that of the tubules preincubated either in NaCl or Na₂SO₄ solutions, an observation suggesting that choline ions enter rat MAL cells. Barium (3 mmol/l) prevented tubular swelling. This inhibition corresponded to a smaller increase in [Na_i] than that observed in control tubules. Furosemide or bumetanide (even at 0.1 mmol/l) did not alter the increases in tubular volume and in Na⁺ content induced by ouabain. The data provide additional evidence that the isoosmotic swelling of MAL cells requires an almost full inhibition of Na⁺-pump activity and involves coupled net fluxes of Na⁺ and Cl^– ions.