[Na+] i /[K+] i -independent death of ouabain-treated renal epithelial cells is not mediated by Na+,K+-ATPase internalization and de novo gene expression

The cytotoxic effect of long-term exposure of renal epithelial cells to ouabain and other cardiotonic steroids (CTS) is mediated by the interaction of these compounds with Na⁺,K⁺-ATPase but is independent of the inhibition of Na⁺,K⁺-ATPase-mediated ion fluxes. Sustained application of CTS also leads to Na⁺,K⁺-ATPase endocytosis and its translocation into the nuclei that might trigger the cell death machinery via the regulation of gene expression. This study examines the role of Na⁺,K⁺-ATPase internalization and de novo gene expression in the death of ouabain-treated C7-Madin–Darby canine kidney (MDCK) cells derived from distal tubules of the MDCK. In these cells, 6-h exposure to 3 μM ouabain led to the internalization of ∼50% of plasmalemmal Na⁺,K⁺-ATPase. Prolonged incubation in a K⁺-free medium abolished ouabain-induced Na⁺,K⁺-ATPase internalization but did not affect the cytotoxic action of ouabain seen after 18-h incubation. Previously, it was shown that CTS-induced Na⁺,K⁺-ATPase internalization is mediated by its interaction with Src within caveolae. Neither caveolae damage by cholesterol depletion with methyl-β-cyclodextrin nor Src inhibition with 4-amino-5(4-chlorophenyl)-7-(t-butyl)pyrazol[3,4-d]pyridine affected the death of ouabain-treated C7-MDCK cells. Actinomycin D at the 0.1-μg/ml concentration almost completely abolished ribonucleic acid synthesis but did not protect C7-MDCK cells from the cytotoxic action of ouabain. Our results show that neither Na⁺,K⁺-ATPase endocytosis nor de novo gene expression contributes to -independent cell death signaling evoked by prolonged exposure to CTS.     

Electrogenic Na+/K+-transport in human endothelial cells

Na⁺/K⁺ pump currents were measured in endothelial cells from human umbilical cord vein using the whole-cell or nystatin-perforated-patch-clamp technique combined with intracellular calcium concentration ([Ca²⁺]_i) measurements with Fura-2/AM. Loading endothelial cells through the patch pipette with 40 mmol/l [Na⁺] did not induce significant changes of [Ca²⁺]_i. Superfusing the cells with K⁺-free solutions also did not significantly affect [Ca²⁺]_i. Reapplication of K⁺ after superfusion of the cells with K⁺-free solution induced an outward current at a holding potential of 0 mV. This current was nearly completely blocked by 100 mol/l dihydroouabain (DHO) and was therefore identified as a Na⁺/K⁺ pump current. During block and reactivation of the Na⁺/K⁺ pump no changes in [Ca²⁺]_i could be observed. Pump currents were blocked concentration dependently by DHO. The concentration for half-maximal inhibition was 21 mol/l. This value is larger than that reported for other tissues and the block was practically irreversible. Insulin (10–1000 U/l) did not affect the pump currents. An increase of the intracellular Na⁺ concentration ([Na⁺]_i) enhanced the amplitude of the pump current. Half-maximal activation of the pump current by [Na⁺]_i occurred at about 60 mmol/l. The concentration for half-maximal activation by extracellular K⁺ was 2.4±1.2 mmol/l, and 0.4±0.1 and 8.7±0.7 mmol/l for Tl⁺ and NH₄ ⁺ respectively. The voltage dependence of the DHO-sensitive current was obtained by applying linear voltage ramps. Its reversal potential was more negative than –150 mV. Pump currents measured with the conventional whole-cell technique were about four times smaller than pump currents recorded with the nystatin-perforated-patch method. If however 100 mol/l guanosine 5-O-(3-thiotriphosphate) (GTPS) were added to the pipette solution, the currents measured in the ruptured-whole-cell-mode were not significantly different from the currents measured with the perforated-patch technique. We suppose that the use of the perforated-patch technique prevents wash out of a guanine nucleotide-binding protein (G-protein)-connected intracellular regulator that is necessary for pump activation.     

Regulation of the cytosolic pH set point for activation of the Na+/H+ antiport in human platelets: the roles of the Na+/Ca2+ exchange,the Na+-K+-2Cl− cotransport and cellular volume

To explore further the mechanisms that regulate the Na⁺/H⁺ antiport in human platelets, we examined the effect of Na⁺ pump inhibition by ouabain and K⁺ removal from the extracellular medium on parameters of this transport system. Treatment with ouabain resulted in increased cytosolic free Ca²⁺ and Na⁺, coupled with an alkaline shift in the cytosolic pH set point for the Na⁺/ H⁺ antiport. Inhibition of the Na⁺ pump by the removal of K⁺ from the medium increased the cytosolic Na⁺ but not the cytosolic Ca²⁺; yet this treatment also produced a substantial alkaline shift in the cytosolic pH set point for the Na⁺/H⁺ antiport. This effect appeared to relate to a decline in cellular volume and it was attenuated by the Na⁺-K⁺-2Cl^– cotransport inhibitor, bumetanide. These findings indicate: (a) a link between the Na⁺ pump and the Na⁺/H⁺ antiport, mediated by the Na⁺/Ca²⁺ exchange and the cytosolic free Ca²⁺, and (b) a link between the Na⁺/H⁺ antiport and the Na⁺-K⁺-2Cl^– cotransport through cellular volume.This work was supported by grants from the National Heart, Lung, and Blood Institute (HL34807, HL42856) and the American Diabetes Association. M. Kimura is a postdoctoral research fellow of the American Heart Association, New Jersey Affiliate     

Intracellular Na+ modulates large conductance Ca2+-activated K+ currents in human umbilical vein endothelial cells

We studied the effects of Na⁺ influx on large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na⁺ replacement by NMDG⁺ or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na⁺ concentration ([Na⁺]_i) and vice versa. In addition, extracellular Na⁺ depletion decreased [Na⁺]_i. In voltage-clamped cells, BK_Ca currents were markedly increased by extracellular Na⁺ depletion. In inside-out patches, increasing [Na⁺]_i from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK_Ca channels and decreasing intracellular K⁺ concentration ([K⁺]_i) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na⁺]_i gradually from 0 to 70 mM, by replacing K⁺, markedly reduced single channel conductance and NPo. The Na⁺–Ca²⁺ exchange blocker Ni²⁺ or KB-R7943 decreased [Na⁺]_i and increased BK_Ca currents simultaneously, and the Na⁺ ionophore monensin completely inhibited BK_Ca currents. BK_Ca currents were significantly augmented by increasing extracellular K⁺ concentration ([K⁺]_o) from 6 to 12 mM and significantly reduced by decreasing [K⁺]_o from 12 or 6 to 0 mM or applying the Na⁺–K⁺ pump inhibitor ouabain. These results suggest that intracellular Na⁺ inhibit single channel conductance of BK_Ca channels and that intracellular K⁺ increases single channel conductance and NPo. GH Liang and MY Kim contributed equally to this publication and therefore share the first authorship.     

The effect of ouabain on intracellular activities of K+, Na+, Cl−, H+ and Ca2+ in proximal tubules of frog kidneys

Using conventional and ion selective microelectrodes, the effect of ouabain (10^–4 mol/l) on peritubular cell membrane potential (PD_pt), on intracellular pH (pH_i) as well as on the intracellular ion activities of Cl^– (Cl _i ^– ), K⁺ (K _i ⁺ ), Na⁺ (Na _i ⁺ ) and Ca²⁺ (Ca _i ²⁺ ) was studied in proximal tubules of the isolated perfused frog kidney. In the absence of ouabain (PD_pt=–57.0±1.9 mV), the electrochemical potential difference of chloride (apparent {ie6-1} and of potassium {ie6-2} is directed from cell to bath, of H⁺ {ie6-3}, of Na⁺ {ie6-4} and of Ca²⁺ {ie6-5} from bath to cell. Ouabain leads to a gradual decline of PD_pt, which is reduced to half (PD_{pt, 1/2}) within 31±4.6 min (in presence of luminal glucose and phenylalanine), and to a decline of the absolute values of apparent {ie6-6}, of {ie6-7}, {ie6-8} and {ie6-9}. In contrast, an increase of {ei6-10} is observed. At PD_{pt, 1/2} apparent Cl _i ^– increases by 6.2±1.0 mmol/l, pH_i by 0.13±0.03, Ca _i ²⁺ by 185±21 nmol/l, and Na _i ⁺ by 34.2±4.6 mmol/l, whereas K _i ⁺ decreases by 37.7±2.2 mmol/l. The results suggest that the application of ouabain is followed by a decrease of peritubular cell membrane permeability to K⁺, by an accumulation of Ca²⁺, Na⁺ and HCO ₃ ^- in the cell and by a dissipation of the electrochemical Cl^– gradient.Supported by Österr. Forschungsrat, Proj. No. 4366     

The Na+/K+ pump of cardiac Purkinje cells is preferentially fuelled by glycolytic ATP production

The role of glycolysis and oxidative phosphorylation in providing the ATP for the cardiac Na⁺/K⁺ pump was studied in cardioballs from sheep Purkinje fibres. As an indicator of the pump activity, the pump current I _p was measured at -20mV and 30–33° C by means of whole-cell recording. During intracellular perfusion with a pipette solution containing 5 mM ATP and 15 mM glucose I _p reached a maximum within 8 min and declined to 50% of this value within 27 min after gaining access to the cell interior. Perfusion with an ATP- and glucose-free medium barely enhanced the I _p decline. Inhibition of the oxidative phosphorylation by carbonylcyanide m-chlorophenylhydrazone (CCCP, 2 M or 20 M) moderately accelerated the effect of the ATP- and glucose-free pipette solution. Addition of 2 mM iodoacetic acid (an inhibitor of glycolysis) to the latter medium further enhanced the I _p decrease with time. Inhibition of the glycolytic ATP synthesis by 2-deoxy-D-glucose (5 mM) caused a dramatic decline of I _p to half of its maximum within 7.3 min. Pyruvate (5 mM) and inorganic phosphate (2 mM) did not affect the fast I _p decline evoked by the ATP- and glucose-free, 2-deoxyglucose-containing medium, whereas 2 M CCCP still hastened the fast I _p decrease slightly. This effect of complete metabolic inhibition was reversed by switching to an inhibitor-free pipette solution containing 15 mM ATP. It is concluded that the Na⁺/K⁺ pump of cardiac Purkinje cells is preferentially fuelled by glycolytic ATP synthesis.This work was supported by the Deutsche Forschungsgemeinschaft (Forschergruppe Konzell)     

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.     

Copper toxicity in cultured human skeletal muscle cells: the involvement of Na+/K+-ATPase and the Na+/Ca2+-exchanger

Copper (Cu²⁺) intoxication has been shown to induce pathological changes in various tissues. The mechanism underlying Cu²⁺ toxicity is still unclear. It has been suggested that the Na⁺/K⁺-ATPase and/or a change of the membrane permeability may be involved. In this study we examined the effects of Cu²⁺ on the Na⁺ and Ca²⁺ homeostasis of cultured human skeletal muscle cells using the ion-selective fluorescent probes Na⁺-binding benzofuran isophtalate (SBFI) and Fura-2, respectively. In addition, we measured the effect of Cu²⁺ on the Na⁺/K⁺-ATPase activity. Cu²⁺ and ouabain increase the cytoplasmic free Na⁺ concentration ([Na⁺]_i). Subsequent addition of Cu²⁺ after ouabain does not affect the rate of [Na⁺]_i increase. Cu²⁺ inhibits the Na⁺/K⁺-ATPase activity with an IC₅₀ of 51 M. The cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) remains unaffected for more than 10 min after the administration of Cu²⁺. Thereafter, [Ca²⁺]_i increases as a result of the Na⁺/Ca²⁺-exchanger operating in the reversed mode. The effects of Cu²⁺ on the Na⁺ homeostasis are reversed by the reducing and chelating agent dithiothreitol and the heavy metal chelator N,N,N,N,-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). In conclusion, SBFI is a good tool to examine Na⁺ homeostasis in cultured human skeletal muscle cells. Under the experimental conditions used, Cu²⁺ does not modify the general membrane permeability, but inhibits the Na⁺/K⁺-pump leading to an increase of [Na⁺]_i. As a consequence the operation mode of the Na⁺/Ca²⁺-exchanger reverses and [Ca²⁺]_i rises.The authors thank staff and coworkers of the Department of Neurology of the University Hospital Nijmegen, Nijmegen for their kind cooperation in obtaining muscle biopsies. Mr. Arie Oosterhof is gratefully acknowledged for culturing of the human muscle cells. The Prinses Beatrix Fonds and the Dutch-Chinese scientific exchange program contributed financial support for this study.     

Relation between extracellular [K+], membrane potential and contraction in rat soleus muscle: modulation by the Na+-K+ pump

An increased extracellular K⁺ concentration ([K⁺]₀) is thought to cause muscle fatigue. We studied the effects of increasing [K⁺]₀ from 4 mM to 8–14 mM on tetanic contractions in isolated bundles of fibres and whole soleus muscles from the rat. Whereas there was little depression of force at a [K⁺]₀ of 8–9 mM, a further small increase in [K⁺]₀ to 11–14 mM resulted in a large reduction of force. Tetanus depression at 11 mM [K⁺]_o was increased when using weaker stimulation pulses and decreased with stronger pulses. Whereas the tetanic force/resting membrane potential (E _M) relation showed only moderate force depression with depolarization from –74 to –62 mV, a large reduction of force occurred whenE _M fell to –53 mV. The implications of these relations to fatigue are discussed. Partial inhibition of the Na⁺-K⁺ pump with ouabain (10^–6 M) caused additional force loss at 11 mM [K⁺]₀. Salbutamol, insulin, or calcitonin gene-related peptide all stimulated the Na⁺-K⁺ pump in muscles exposed to 11 mM [K⁺ ₀] and induced an average 26–33% recovery of tetanic force. When using stimulation pulses of 0.1 ms, instead of the standard 1.0-ms pulses, force recovery with these agents was 41–44% which was significantly greater (P < 0.025). Only salbutamol caused any recovery ofE _M (1.3 mV). The observations suggest that the increased Na⁺ concentration difference across the sarcolemma, following Na⁺-K⁺ pump stimulation, has an important role in restoring excitability and force.     

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.     

O2 consumption,aerobic glycolysis and tissue phosphagen content during activation of the NA+/K+ pump in rat portal vein

Oxygen consumption, lactate production and tissue contents of ATP, phosphocreatine (PCr) and lactate were measured following readdition of K⁺ to K⁺-depleted rat portal veins, in order to study the energy turnover associated with Na⁺/K⁺ pumping. During incubation in K⁺-free medium at 37° C spontaneous contractions disappeared in 10–20 min. Readdition of K⁺ (5.9 mM) after 40 min K⁺-free incubation caused hyperpolarization of the cell membrane for the first 5–10 min and then gradual depolarization with return of spontaneous action potentials and contractions by 10–20 min. During the first 4–6 min after K⁺ readdition aerobic lactate production was about doubled and then gradually returned to the original level (0.17 mol/min g) at about 20 min. The increase in glycolytic rate was prevented by 1 mM ouabain. In contrast, O₂ consumption (in K⁺-free medium, 0.38 mol/min g) rose by about 10% when K⁺ was added and this increase lasted about 5 min. By 8 min after K⁺ addition the increased glycolysis and oxidative phosphorylation had accounted for each about the same amount of extra ATP generation over that extrapolated from the steady rate before K⁺ addition. The average total increase in ATP turnover in the first 8 min was 15%. During this period there was no change in the cellular content of ATP, PCr, or extractable ADP. The results indicate that Na⁺/K⁺ pumping utilizes a relatively small share of the total energy turnover in the vascular smooth muscle but is to a large extent dependent on aerobic glycolysis and therefore a major site of carbohydrate usage.     

Inhibition of voltage-dependent Na+ and K+ currents by forskolin in nodes of Ranvier

The effect of forskolin on voltage-activated Na⁺ and K⁺ currents in nodes of Ranvier from the toad, Bufo marinus, has been examined using the vaseline-gap voltageclamp technique. Peak Na⁺ currents (I _Na) were reduced by 35% and the rate of decline of Na⁺ current during continuous depolarization was accelerated following treatment with 450 M forskolin. However, the voltage-dependence of steady-state inactivation as well as the rate of recovery from fast inactivation remained unchanged. Upon repetitive depolarization at 1–10 Hz, a further inhibition of I _Na (60%) was observed. This use-dependent or phasic inhibition recovers slowly at -80 mV ( 13 s) and had a voltage-dependence like that of activation of the Na conductance. Near maximal steady-state phasic inhibition occurred with depolarizing pulse durations of only 4 ms, consistent with a direct involvement of the open Na⁺ channel in the blocking process. Inhibition of the delayed K⁺ current (I _K) was characterized by a concentration-dependent reduction in steady-state current amplitude (IC₅₀ 80 M) and a concentration-independent acceleration of current inactivation. A similar inhibition of I _K was obtained with 1,9-dideoxyforskolin, a homolog which does not activate adenylate cyclase (AC). The results suggest that the inhibition of I _K and perhaps I _Na follows directly from drug binding and is not a consequence of AC activation.     

Human colonic (Na++K+)-ATPase and specific ouabain binding are not influenced by lipoxygenase products or superoxide radicals

Summary The effects of lipoxygenase products (5-, 12-, 15-HETE, LTB₄) and superoxide radicals on human colonic (Na⁺+K⁺)-ATPase and specific ouabain binding were measured. No significant inhibition in concentrations up to 3 × 10^–5 M was observed. The results are discussed with regard to a possible role of lipoxygenase products and radicals in the pathogenesis of water and electrolyte disturbances in various diarrheal states including inflammatory bowel disease.Abbrevations IBD Inflammatory bowel disease - HETE Hydroxytetraenoic acid - LTB₄ Leukotriene B₄ Supported by DFG (Er 65/4-4)     

Construction of K+- and Na+-sensitive theta-microelectrodes with fine tips: an easy method with high yield

A new method is described to prepare theta-microelectrodes with tips up to 0.15 m diameter controlled under scanning electron microscope. K⁺- and Na⁺-sensitive resins were tested.Method features are the following: i) hard drying of the glass, ii) rehydration of one channel and weak wetting of the other with a three-methylchlorosilane solution before pulling, iii) simultaneous presence of water and silane in the two channels during pulling, iv) gradual silanization from the tip to the shank.Selective and conventional channels did not affect each other and no displacements of resins were observed. The change of potential difference of the selective channel was more than –50 mV/decade. Apical membrane potentials and cell Na⁺ and K⁺ activities of the epithelial cells of rabbit gall-bladder (cell diameter: 5–10 m) were measured with these theta-microelectrodes and with single-barrel microelectrodes of similar tip size: results obtained were not significantly different.     

Activation of the Na+/K+-ATPase by insulin and glucose as a putative negative feedback mechanism in pancreatic beta-cells

Pancreatic beta-cells of sulfonylurea receptor type 1 knock-out (SUR1(-/-)) mice exhibit an oscillating membrane potential (V (m)) demonstrating that hyper-polarisation occurs despite the lack of K(ATP) channels. We hypothesize that glucose activates the Na(+)/K(+)-ATPase thus increasing a hyper-polarising current. Elevating glucose in SUR1(-/-) beta-cells resulted in a transient fall in V (m) and [Ca(2+)](c) independent of sarcoplasmic and endoplasmic reticulum Ca(2+)-activated ATPase (SERCA) activation. This was not affected by K(+) channel blockade but inhibited by ATP depletion and by ouabain. Increasing glucose also reduced [Na(+)](c), an effect reversed by ouabain. Exogenously applied insulin decreased [Na(+)](c) and hyper-polarised V (m). Inhibiting insulin signalling in SUR1(-/-) beta-cells blunted the glucose-induced decrease of [Ca(2+)](c). Tolbutamide (1 mmol/l) disclosed the SERCA-independent effect of glucose on [Ca(2+)](c) in wild-type beta-cells. The data show that in SUR1(-/-) beta-cells, glucose activates the Na(+)/K(+)-ATPase presumably by increasing [ATP](c). Insulin can also stimulate the pump and potentiate the effect of glucose. Pathways involving the pump may thus serve as potential drug targets in certain metabolic disorders.     

Roles of K+ channels in regulating tumour cell proliferation and apoptosis

K⁺ channels are a most diverse class of ion channels in the cytoplasmic membrane and are distributed widely in a variety of cells including cancer cells. Cell proliferation and apoptosis (programmed cell death or cell suicide) are two counterparts that share the responsibility for maintaining normal tissue homeostasis. Evidence has been accumulating from fundamental studies indicating that tumour cells possess various types of K⁺ channels, and that these K⁺ channels play important roles in regulating tumour cell proliferation and apoptosis, i.e. facilitating unlimited growth and promoting apoptotic death of tumour cells. The potential implications of K⁺ channels as a pharmacological target for cancer therapy and a biomarker for diagnosis of carcinogenesis are attracting increasing interest. This review aims to provide a comprehensive overview of current status of research on K⁺ channels/currents in tumour cells. Focus is placed on the roles of K⁺ channels/currents in regulating tumour cell proliferation and apoptosis. The possible mechanisms by which K⁺ channels affect tumour cell growth and death are discussed. Speculations are also made on the potential implications of regulation of tumour cell proliferation and apoptosis by K⁺ channels.     

Fluorescence and Laser Photon Counting: Measurements of Epithelial [Ca2+]i or [Na+]i with Ciliary Beat Frequency

We describe a system we developed that enabled simultaneous measurements of either epithelial calcium ion concentration ([Ca²⁺]i) or sodium ion concentration [Na⁺]i with the ciliary beat frequency (CBF) in native ciliated epithelia using either Fura-2 (AM) or SBFI (AM) ratiometric fluorescence photon counting along with nonstationary laser light scattering. Studies were performed using native epithelial tissues obtained from ovine tracheae. The dynamic range of the laser light-scattering system was determined by a simulated light beating experiment. The nonstationary CBF was demonstrated by the time-frequency analysis of the raw photon count sequences of backscattered heterodyne photons from cultured and native epithelia. Calibrations of calcium and sodium ion concentrations were performed using the respective Fura-2 and SBFI impermanent salts as well as in native epithelia. The cumulative responses of 10^–6, 10^–5, and 10^–4} M nifedipine on [Ca²⁺]i together with the CBF as well as the cumulative responses of 10^–5, 10^–4, and 10^–3 M amiloride on [Na⁺]i together with the CBF were also determined. Nifedipine decreased [Ca²⁺]i but had no effect on CBF. Amiloride decreased [Na⁺]i and CBF. Stimulation of CBF corresponded with either an increase of [Na⁺]i or an increase of [Ca²⁺]i. Decreases of [Na⁺]i or substantial decreases of [Ca²⁺]i were associated with decreases in the CBF. These data demonstrate the utility of this system for investigating the regulatory mechanisms of intracellular ions dynamics and the CBF in native epithelia. © 1998 Biomedical Engineering Society. PAC98: 8780+s, 8722-q, 4262Be     

Influence of the external K+-concentration on the electrical activity and the Na+-, K+-content of the retinal tissue

Summary The effect of the external K⁺-concentration in a range from 0 to 10 mMol/l on the exposure potential (ERG) and the Na⁺-, K⁺-distribution in the retinal tissue of Rana esculenta was investigated. After 45 min of perfusion with the test solution moist combustion of the tissue and analysis by flame photometry was carried out.At concentrations of less than 1 mMol/l an extensive loss of retinal potassium could be observed which corresponded to a slow and steady decrease of the exposure potential. Simultaneously the mean intracellular Na⁺-content increased. Above 2 mMol/l a rapid decline of the potential _b to a constant level was recorded. At the same time there was a slight increase in retinal potassium and a decrease in sodium.The quick transition to steady values of the ERG at high external K⁺-concentrations is attributed to the limitation of the potassium uptake of retinal cells which brings about a constant distribution of electrolytes in a short time. The slow drop in potential at a low potassium content of the bathing solution is ascribed to the diffusion process of K⁺ through cellular membranes of the retina.Partly supported by the Deutsche Forschungsgemeinschaft.     

The colon carcinoma cell line Caco-2 contains an H+/K+-ATPase that contributes to intracellular pH regulation

The presence of an H⁺/K⁺-ATPase and its contribution to the regulation of intracellular pH (pH_i) was investigated in Caco-2 cells. The H⁺/K⁺-ATPase was detected immunologically using the monoclonal antibody 5-B6, which was raised against hog gastric H⁺/K⁺-ATPase. Cell pH was determined using the pH-sensitive dye 2,7-bis(carboxyethyl)-carboxyfruorescein. Control pH_i, measured in HCO ₃ ^– -free medium, was 7.62±0.03 (n=27) when cells were cultured for 14 days and decreased to 7.40±0.03 (n=18) after 35 days in culture. Recovery of pH_i following a NH ₄ ⁺ /NH₃ pulse could be reduced by either 100 M SCH 28080 or 1 mM amiloride, or by removing extracellular Na⁺. The inhibitory effects of SCH 28080 and amiloride were additive, demonstrating the involvement of a gastric-like H⁺/K⁺-ATPase and a Na⁺/H⁺ exchanger in regulating pH_i. Recovery rates at pH_i 6.8 were not significantly different in cells cultured for up to 21 days, but were significantly lower in cells cultured for 28 and 35 days. This decrease in recovery rate was due to a decrease in the SCH-28080-insensitive recovery, indicating a reduction of the relative importance of Na⁺/H⁺ exchange to the recovery. Recovery of pH_i was also inhibited by 1 mM N-ethylmaleimide. However, it is unlikely that N-ethylmaleimide inhibited a vacuolar type of H⁺-ATPase, since bafilomycin A₁ had no effect on pH_i recovery. In conclusion, Caco-2 cells contain a SCH-28080-sensitive mechanism for regulating pH_i, which is most conveniently studied after 28 days in culture, when the relative contribution of a Na⁺/H⁺ exchanger to pH_i regulation is decreased.