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.     

Potassium and sodium shifts during in vitro isometric muscle contraction,and the time course of the ion-gradient recovery

Intracellular potassium ([K⁺]_i), interstitial potassium ([K⁺]_inter), intracellular sodium ([Na⁺]_i), and resting membrane potential (RMP) were measured before and after repetitive stimulation of mouse soleus and EDL (extensor digitorum longus) muscles. At rest, RMP was –69.8 mV for soleus and –74.9 mV for EDL (37°C). [K⁺]_i was 168 mM and 182 mM, respectively. In soleus, free [Na⁺]_i was 12.7 mM. After repetitive stimulation (960 stimuli) RMP had decreased by 11.9 mV for soleus and by 18.2 mV for EDL. [K⁺]_i was reduced by 32 mM and 48 mM, respectively, whereas [K⁺]_inter was doubled. In soleus [Na⁺]_i had increased by 10.6 mM, demonstrating that the [K⁺]_i-decrease is three times higher than the [Na⁺]_i-increase. It is concluded that this difference reflects different activity induced movements of Na and K, and that the difference is not due to the Na/K pumping ratio. The possible involvement of the potassium loss in muscle fatigue is discussed. After stimulation RMP recovered with a time constant of 0.9 min for soleus and 1.5 min for EDL. Within the first minutes after stimulation the intracellular potassium concentration increased by 20.4 mM/min for soleus and 21.7 mM/min for EDL. Free [Na⁺]_i decreased with less than 10 mM/min. The mechanisms underlying the different rate of changes are discussed.Parts of this work have been published in preliminary form (Juel and Sjøgaard 1984)     

Na+-Ca2+ exchange in the isolated cochlear outer hair cells of the guinea-pig studied by fluorescence image microscopy

The outer hair cell isolated from the guinea-pig was superfused in vitro and the cytosolic calcium concentration ([Ca²⁺]_i) and sodium concentration ([Na⁺]_i) were measured using fluorescence indicators. Under the resting condition, [Ca²⁺]_i and [Na⁺]_i were 91±9 nM (n = 51) and 110±5 mM (n = 12), respectively. Removal of external Na⁺ by replacing with N-methyl-D-glucamine (NMDG⁺) increased [Ca²⁺]_i by 270±79% (n = 27) and decreased [Na⁺]_i by 23±4 mM (n = 6). Both changes in [Ca²⁺]_i and [Na⁺]_i were totally reversible on returning external Na⁺ to the initial value and were inhibited by addition of 0.1 mM La³⁺ or 100 M amiloride 5-(N,N-dimethyl) hydrochloride. Elevation of external Ca²⁺ ions to 20 mM reversibly decreased [Na⁺]_i by 8±6 mM (n = 5). Moreover, the chelation of the intracellular Ca²⁺ with 1,2-bis (2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA) exerted an inhibitory action on the NMDG⁺-induced reduction in [Na⁺]_i. Exposure to 5 mM NaCN for 2 min significantly and reversibly increased [Ca²⁺]_i by 290±37% (n = 5), but did not affect the [Ca²⁺]_i elevation induced by the NMDG⁺ solution. The rise in [Ca²⁺]_i induced by the NMDG⁺ solution was not enhanced by ouabain pretreatment. Addition of ouabain did not alter the [Na⁺]_i. The present results are best explained by the presence of an Na⁺-Ca²⁺ exchanger in cell membrane and indicate that the activity of Na⁺/K⁺ pump is poor in outer hair cells.     

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.     

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.     

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.     

Na+-activated K+ current in cardiac cells: rectification,open probability,block and role in digitalis toxicity

The Na⁺-activated K⁺ current was studied in inside-out patches and in whole cells isolated from the guinea-pig cardiac ventricle. The single channel conductance showed inward rectification for K⁺ _i+ _e, but outward rectification for K⁺ _i>K⁺ _e The open probability was dependent on Na⁺ _i and Na⁺,K⁺-pump activity. In the presence of pump blockade the channel remained active at low Na⁺ _i Similar results were obtained in whole cells. These results suggest the existence of Na⁺ gradients depending on Na⁺,K⁺-pump activity and passive inward leak of Na⁺. The channel and whole cell current were blocked by R56865. The drug did not change the single channel conductance but markedly reduced open probability by shortening burst duration. The current may play an important role in action potential shortening during pump blockade.This work was supported by a grant of the National Fund for Scientific Research Belgium.3.0016.87.     

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     

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.     

Changes in extracellular potassium and calcium in rat cerebellar cortex related to local inhibition of the sodium pump

Extracellular K⁺, Ca²⁺, and Na⁺ ([K⁺]_e, [Ca²⁺]_e, [Na⁺]_e) were recorded with ion selective microelectrodes in the cerebellar cortex of urethane-anesthetized rats. Superfusion of the cerebellum with artificial cerebrospinal fluid containing K-strophanthidin (10^–6–10^–4 mol/l) or other cardioactive steroids, known to be inhibitors of the sodium/potassium pump, had the following effects: elevation of resting [K⁺]₃, reduction of poststimulus K⁺-undershoots, decrease of resting [Ca²⁺]_e and [Na⁺]_e. For instance, at 3×10^–5 mol/l K-strophanthidin within the superfusion solution (the unknown intracerebellar concentration being certainly much smaller), [K⁺]_e was elevated up to 130% and [Ca²⁺]_e reduced to 70% of their resting values. Iontophoretic K⁺-pulses were enhanced in amplitude at the same time. Control experiments with iontophoretic TMA application demonstrated that the glycoside effects were not due (or in higher concentrations only partly due) to shrinkage of the extracellular fluid volume. When tetrodotoxin (10^–7 mol/l) or Mn²⁺ (1–3 mmol/l) were additionally superfused, K-strophanthidin effects were qualitatively similar, though quantitatively smaller. This indicates that part of the effects were indirect via neuronal activity evoked by the blockade of the sodium pump. The experiments show that reduction of sodium pump activity in cerebellar cortex has rapid and serious consequences on the distribution of potassium and calcium in the extracellular space, resulting in an alteration of neuronal circuit excitability.     

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.     

Sodium movement into and out of corneal endothelium

Rabbit corneal endothelial cells mounted in vitro were impaled simultaneously with Na⁺-selective and conventional KCl-filled microelectrodes. The membrane potential (V _m) was –30.4±0.8 mV (mean ±SEM, n = 55) and the intracellular [Na⁺]_i (calculated from the Na⁺-selective electrode potential, V_Na) was 13.7 ±1.9 mM (mean±SEM, n = 16). When ouabain was added to the perfusate the cell depolarised, causing both V _m and V_Na to increase with a very similar time course. Final V _m was –6.3±0.6 mV (mean ±SEM, n = 15), and the final [Na⁺]_i was 114±6.9 mM (mean ± SEM, n = 5). The parallel increase in V _m and rise in [Na⁺]_i suggest that a component of the ouabain-induced depolarisation of the cell (increase in V _m) is due to Na⁺ entry into the cell down its concentration gradient. The lateral and basal location of the Na⁺/K⁺-ATPase in bovine endothelial cells was confirmed (for the first time at the electron-microscopic level) using a monoclonal antibody specific for the 1 subunit of Na⁺/K⁺-ATPase. The absence of a net Na⁺ flux across these cells combined with the basolateral location of the ATPase suggest that Na⁺ exit from the cell, and its re-entry take place across the same membrane (i. e. the basolateral).     

Changes in intracellular ion activities induced by adrenaline in human and rat skeletal muscle

To study the stimulating effect of adrenaline (ADR) on active Na⁺/K⁺ transport we used double-barrelled ion-sensitive micro-electrodes to measure the activities of extracellular K⁺ (aK_e) and intracellular Na⁺ (aNa_i) in isolated preparations of rat soleus muscle, normal human intercostal muscle and one case of hyperkalemic periodic paralysis (h.p.p.). In these preparations bath-application of ADR (10⁻⁶ M) resulted in a membrane hyperpolarization and transient decreasesaK_e andaNa_i which could be blocked by ouabain (3×10⁻⁴ M). In the h.p.p. muslce a continuous rise ofaNa_i induced by elevation ofaK_e to 5.2 mM could be stopped by ADR. In addition, the intracellular K⁺ activity (aK_i), the free intracellular Ca²⁺ concentration (pCa_i) and intracellular pH (pH_i) were monitored in rat soleus muscle. During ADRaK_i increased, pH_i remained constant and intracellular Ca²⁺ apparently decreased. In conclusion, our data show that ADR primarily stimulates the Na⁺/K⁺ pump in mammalian skeletal muscle. This stimulating action is not impaired in the h.p.p. muscle. Parts of the results have been presented to the German Physiological Society (Ballanyi and Grafe 1987)     

Na/Ca exchangers in collecting cells of rat kidney

Single pieces of fura-2-loaded cortical collecting tubule (CCT) isolated either from normal or adrenalectomized (ADX) rats were superfused in vitro, and the cytosolic calcium concentration ([Ca²⁺]_i) was calculated from fluorescence recordings. The effects of altering the sodium gradient across cell membranes were investigated. Switching external sodium from 164 mM to 27 mM (low [Na⁺]_o) had little effect on [Ca²⁺]_i in normal tubules (106±9 versus 101±9 nM, n=15) whereas it resulted in a large peak of [Ca²⁺]_i in CCT from ADX-rats (270±32 versus 135±11 nM, n=21). Since CCT from ADX rats are known to have a reduced Na-pump activity, the effect of ouabain treatment on CCT from normal rats was also tested. When CCT from normal rats were exposed to 1 mM of ouabain in the presence of 164 mM of [Na⁺]_o, [Ca²⁺]_i increased only moderately (123±15 versus 111±11 nM, n=13); when the low [Na⁺]_o solution was applied to these ouabain-treated tubules, a large and transient increase in [Ca²⁺]_i was obtained (287±38 versus 123±15 nM, n=13). This response was absent with [Ca²⁺]_o=0. The data suggest the presence of 3 Na⁺/1 Ca²⁺ exchangers in cell membranes of rat CCT. The calcium flux equation derived by Läuger for the exchanger indicates a non-linear relationship between net calcium flux and driving force which could account for the difference observed here between the poor effect of applying either low [Na⁺]_o or ouabain alone and the large peak of [Ca²⁺]_i induced by combining these two conditions.     

Über das Membranpotential des Meerschweinchenvorhofes nach Hypothermie

Zusammenfassung An isolierten Vorhöfen des Meerschweinchenherzens wurden der Extracellulärraum (ER), die intracellulären Na⁺-und K⁺-Konzentrationen ([Na_i], [K]_i) und das membranpotential (MP) vor, während und nach Unterkühlung der Präparate gemessen. Das Wiedererwärmen der Vorhöfe erfolgte bei 35° C in Tyrode-Lösungen mit einem K⁺-Gehalt ([K]_e) von 0–21,6 mM/l [K]_e.Der Extracellulärraum (ER) wurde als Inulinraum gemessen. Bei 35° C betrug er etwa 350 ml/kg, bei 4–6° C etwa 300 ml/kg.Der intracelluläre K⁺-Gehalt der Vorhöfe sank während der Unterkühlung ab und nahm bei Wiedererwärmen der Präparate zu. Die intracelluläre Na⁺-Konzentration stieg während der Hypothermie an und fiel während des Wiedererwärmens ab.Das Membranpotential nahm bei Unterkühlung der Vorhöfe ab. Wiedererwärmen verursachte einen raschen Anstieg des Membranpotentiales. In Tyrodelösung mit 21,6 bzw. 10,8 mM/l [K]_e war das Membranpotential innerhalb der ersten 10 min des Wiedererwärmens signifikant mehr negativ als das entsprechende K⁺-Gleichgewichtspotential (E_K). Bei Weidererwärmen der Vorhöfe in einer Tyrodelösung mit 5,4 mM/l [K]_e waren Membranpotential und K⁺-Gleichgewichtspotential in den ersten 10 min gleich hoch. In K⁺-freier Tyrodelösung war das Ansteigen, des Membranpotentiales bei Wiedererwärmen nicht von einer gleichzeitigen Veränderung von [Na]_i begleitet. Unter diesen Bedingungen erreichte das Membranpotential wahrscheinlich nicht die Höhe des K⁺-Gleichgewichtspotentiales.Aus den Versuchsergebnissen wird geschlossen, daß das Membranpotential des Meerschweinchenvorhofes nach Hypothermie von einer elektrogenen Na⁺-Pumpe mit bestimmt werden kann. Die Leistung des elektrogenen Na⁺-Transportes ist von [K]_e und wahrscheinlich von [Na]_i abhängig.     

Effect of sodium concentration and of atropine on the contractile response of the guinea-pig ileum to potassium ions

The possibility that the guinea-pig ileum's contractile response to K⁺-rich solutions is partly mediated by acetylcholine release from the intramural nervous tissue was examined by studying the inhibition of that response by atropine at different values of [Na⁺]₀. In a medium in which the NaCl was replaced by iso-osmotic glucose, the response to high [K⁺]₀ was not greatly affected, while the responses to acetylcholine and to other agonists were significantly reduced. In control medium (149 mM Na⁺), atropine (10^–7 M) partly inhibited the responses to K⁺-rich solutions and to agonists such as histamine, 5-hydroxytryptamine and bradykinin. When [Na⁺]₀ was reduced to 12 mM, by iso-osmotic substitution of glucose for NaCl, the response to high K⁺ was no longer inhibited by atropine, which still partly inhibited the contractions elicited by the three agonists and totally blocked the response to acetylcholine. It is proposed that atropine's inhibition of the response to high K⁺ and to agonists is not due to its specific anti-muscarinic effect, but to an unspecific action, which in the case of the agonists is independent of [Na⁺]₀. In addition, the inhibition of the response to high K⁺ would result from a different Na⁺-dependent mechanism, possibly involving the stimulation of the Na-K pump by atropine. This is supported by the observation that this drug partly relaxed ileum preparations that were contracted by ouabain.     

High selectivity of the i f channel to Na+ and K+ in rabbit isolated sinoatrial node cells

The ionic selectivity of the hyperpolarizationactivated inward current (i _f) channel to monovalent cations was investigated in single isolated sinoatrial node cells of the rabbit using the whole-cell patch-clamp technique. With a 140 mM K⁺ pipette, replacement of 90% external Na⁺ by Li⁺ caused a –24.5 mV shift of the fully activated current/voltage I/V curve without a significant decrease of the slope conductance. With a 140 mM Cs⁺ pipette, the i _f current decreased almost proportionally to the decrease in external [Na⁺]_o as Li⁺ was substituted. These responses are practically the same as those observed with N-methyl glucamine (NMG⁺) substitution, suggesting that the relative permeability of Li⁺ compared with Na⁺ for the i _f channel is as low as that of NMG⁺. When Cs⁺ or Rb⁺ was substituted for internal K⁺, the fully activated I/V relationship for i _f showed strong inward rectification with a positive reversal potential, indicating low permeability of the i _f channel for Cs⁺ and Rb⁺. These results show that the i _f channel is highly selective for Na⁺ and K⁺ and will not pass the similar ions Li⁺ and Rb⁺. Such a high degree of selectivity is unique and may imply that the structure of the i _f channel differs greatly from that of other Na⁺ and K⁺ conducting channels.     

Potassium-induced relaxation in isolated cerebral arteries contracted with prostaglandin F2α

Summary In helically cut strips of canine cerebral arteries exposed to 5.4 mM [K⁺]_o and contracted with prostaglandin F₂, the addition of K⁺ in concentrations ranging from 0.5–5 mM caused a dose-related relaxation. The relaxing effect of K⁺ was potentiated at reduced [K⁺]_o and suppressed at reduced [Na⁺]_o. Reduction of Cl^– from bathing media failed to alter the effect of K⁺. Removal of external Ca²⁺ markedly attenuated the K⁺-induced relaxation and increase in [Ca²⁺]_o also attenuated the relaxation. Similar relaxation was induced by K⁺ in cerebral arteries from other species including humans, puppies, cats and rabbits. The addition of K⁺ also elicited a relaxation in peripheral arteries, including coronary, femoral, mesenteric and renal, contracted with prostaglandin, but this relaxation was markedly less than in cerebral arteries. The content of Na⁺ in freshly excised cerebral arteries was significantly greater than that of peripheral arteries, while the content of K⁺ in these arteries was not significantly different.The present study provides further evidence to support the hypothesis that an electrogenic Na⁺ pump is involved in the genesis of K⁺-induced relaxation. The Na⁺ pump does not appear to be fully activated at normal [K⁺]_o of 5.4 mM in cerebral arteries.     

Sodium-dependent recovery of ionised magnesium concentration following magnesium load in rat heart myocytes

Our objectives were to investigate regulation of intracellular ionised Mg²⁺ concentration ([fMg²⁺]_i) in cardiac muscle and cardiac Na⁺/Mg²⁺ antiport stoichiometry. [fMg²⁺]_i was measured at 37°C in isolated rat ventricular myocytes with mag-fura-2. Superfusion of myocytes with Na⁺ and Ca²⁺ free solutions containing 30 mM Mg²⁺ for 15 min more than doubled [fMg²⁺]_i from its basal level (0.75 mM). Re-addition of Na⁺ caused [fMg²⁺]_i to fall exponentially with time to basal level, the rate increasing linearly with [Na⁺]. Log(recovery rate) increased linearly with log([Na⁺]), the slope of 1.06 (95% confidence limits, 0.94–1.17) suggesting one Na⁺ ion is exchanged for each Mg²⁺. [fMg²⁺]_i recovery was complete even if the membrane potential was depolarised to 0 mV or if superfusate [Mg²⁺] was increased to 3 mM. Recovery was rapid in normal Tyrode (0.3 min^–1) with a Q₁₀ of 2.2. It was completely inhibited by 200 M imipramine but was unaffected by 20 M KB-R7943 or 1 M SEA0400, suggesting the Na⁺ /Ca²⁺ antiporter is not involved. Membrane depolarisation by increasing superfusate [K⁺] to 70 mM, or voltage clamp to 0 mV, increased recovery rate in Na⁺ containing solutions more than threefold. We conclude [fMg²⁺]_i recovery is by Mg²⁺ efflux on a 1 Na⁺:1 Mg²⁺ antiport.     

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.