The relationship between plasma potassium concentration and muscle torque during recovery following intense exercise

The present study investigated the relationship between plasma potassium ion concentration ([K⁺]) and skeletal muscle torque during three different 15-min recovery periods after fatigue induced by four 30-s sprints. Four males and one female completed the multiple sprint exercise on three separate days; recovery was passive, i.e. no cycling exercise (PRec), active cycling at 30% peak oxygen consumption O_2peak (30% Rec) and active cycling at 60% O_2peak (60% Rec). Plasma [K⁺] was measured from blood sampled from an antecubital vein of subjects at rest and at 0, 3, 5, 10 and 15 min into each recovery. Isokinetic leg strength was measured at rest and at 1, 6, 11 and 16 min during each recovery. Following the exhaustive sprints, [K⁺] increased significantly from an average mean (SEM) resting value of 3.81 (0.07) mmol · l⁻¹ to 4.48 (0.19) mmol · l⁻¹ (P < 0.01). In all recovery conditions, plasma [K⁺] returned to resting levels within 3 min following the fourth sprint. However, in the two active recovery conditions plasma [K⁺] increased over the remainder of the recovery periods to 4.36 (0.12) mmol · l⁻¹ in the 30% Rec condition and 4.62 (0.12) mmol · l⁻¹ in the 60% Rec condition, the latter being significantly higher than the former (P < 0.01). The maximum torque measured following the sprints decreased significantly, on average, to 61.1 (8.36)% of peak levels (P < 0.01). After 15 min of recovery, maximum torque was highest in the 30% Rec condition at 92.13 (3.06)% of peak levels (P < 0.01), compared to 85.23 (3.64)% and 85.71 (0.82)% for the PRec and 60% Rec conditions, respectively. In contrast to the significant differences in plasma [K⁺] across all three recovery conditions, muscle torque recovery was significantly different in only the 30% Rec condition. In summary, recovery of peak levels of muscle torque following fatiguing exercise does not appear to follow changes in plasma [K⁺]. Accepted: 18 October 1996     

Brief antecedent anoxia preserves mitochondrial function after sustained undersupply: A subcellular correlate to ischemic preconditioning?

Background: There is increasing evidence that mitochondria – owning a high degree of autonomy within the cell – might represent the target organelles of the myocardial protection afforded by ischemic preconditioning. It was the aim of the study to investigate a possible subcellular correlate to ischemic preconditioning at the mitochondrial level. In addition, we tested whether this protection depends on mitochondrial ATP-dependent potassium channels (K _ATP) and an might involve an attenuation of mitochondrial ATP hydrolysis during sustained anoxia.Methods and Results: Sustained anoxia (A, 14 min) and reoxygenation (R) completely inhibited state 3 and state 4 respiration of isolated ventricular mitochondria from Wistar rats. An antecedent brief anoxic incubation (4 min) followed by reoxygenation (2 min) prevented this loss of mitochondrial function. The protection afforded by anoxic preconditioning could be mimicked by the K _ATP opener diazoxide (30 μmol/l) and was completely inhibited by the K _ATP blocker 5-hydroxydecanoic acid (300 μmol/l). Structural mitochondrial integrity, as estimated from externalization of the mitochondrial enzymes creatine kinase and glutamateoxalacetate transaminase, remained unchanged between the groups, as did mitochondrial ATP loss during anoxia.Conclusion: For the first time, we provide direct evidence for a subcellular preconditioning-like functional mitochondrial adaptation to sustained anoxia. This effect apparently depends on opening of K_ATP but is independent of ATP preservation.     

Differential effects of glycolytic and oxidative metabolism blockers on the Na-K pump in erythrocytes of the frog, Rana temporaria

This study was undertaken to evaluate the effects of various metabolic blockers on the Na-K-pump activity and ATP content of frog erythrocytes. To eliminate K-C1 cotransport, the frog erythrocytes were incubated in nitrate media at 20 °C. Incubation of the red cells in a glucose-free medium for 2 h had no effect on cell ATP content and K⁺ influx measured as ⁸⁶Rb uptake for 60 min. The Na⁺-K⁺-pump activity was also unchanged in the frog erythrocytes incubated in a glucose-free medium containing 10 mM 2-deoxy-D-glucose or adenosine. Unexpectedly, the treatment of red cells with 1–2 mM glycolytic blocker iodoacetate produced a 2-fold increase in the ouabain-sensitive K⁺ influx. The cell ATP content declined by 9.4% after 2 h of cell incubation with iodoacetate. Incubation of the red cells for 90 min in the presence of 2 mM cyanide, 0.01 mM antimycin A or 5 mM azide resulted in a significant reduction in K⁺ influx by about 50%, 45% and 32%, respectively. The cell ATP content diminished over 60 min and 120 min of cell incubation with 2 mM cyanide by 15.6% and 31.7% of control levels, respectively. In time-course experiments, a 50% reduction in the K⁺ influx was observed when the frog erythrocytes were incubated for only 30 min in the presence of 2 mM cyanide. In contrast, 0.01–0.10 mM rotenone, a site I inhibitor, and 0.01 mM carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of oxidative phosphorylation were without effect on K⁺ influx into frog erythrocytes. These results indicate that about one-half of the Na⁺ -K⁺-pump activity in frog erythrocytes is tightly functionally coupled to cytochromes via a separate “membrane-associated” ATP pool. Accepted: 12 July 1997     

Muscle Potassium and Potassium Losses During Hypokinesia in Healthy Subjects

Hypokinesia (HK) induces electrolyte losses in electrolyte-deficient tissue, yet the mechanisms of electrolyte losses in electrolyte-deficient tissue remain unknown. Mechanisms of electrolyte deposition could be involved. To determine the effect of prolonged HK on potassium (K⁺) deposition were measured muscle K⁺ content and K⁺ losses. Studies were conducted on 20 physically healthy male volunteers during 30 days pre-experimental period and 364 days experimental period. Subjects were equally divided into two groups: control subjects (CS) and experimental subjects (ES). The CS group was run average distances of 9.8 ± 1.7 km day⁻¹ and the ES group was walked average distances of 2.7 ± 0.6 km day⁻¹. Muscle K⁺ content decreased (p < 0.05) and plasma K⁺ concentration, and K⁺ losses in urine and feces increased (p < 0.05) in the ES group compared to their pre-experimental level and the values in their respective CS group. Muscle K⁺ content, plasma K⁺ level, and urine and fecal K⁺ losses did not show any changes in the CS group compared to their pre-experimental values. The conclusion was that K⁺ losses in K⁺-deficient muscle of healthy subjects could have been attributable to the less efficient K⁺ deposition inherently to prolonged HK.     

Resistance to freshwater exposure in White Sea Littorina spp. I: Anaerobic metabolism and energetics

 Anaerobic metabolism and changes in the osmotic concentration of extravisceral fluid were studied in the White Sea periwinkles (Littorina littorea, Littorina saxatilis and Littorina obtusata) during freshwater exposure. Resistance to hypoosmotic stress increased in the order: L. obtusata < L. saxatilis < L. littorea. Our data suggest that osmotic shock is not a primary reason for mortality of the periwinkles under these conditions. During environmental anaerobiosis, considerable succinate accumulation (up to 10–19 μmol g⁻¹ wet weight), and depletion of phosphagen and ATP pools were found in the studied species. Other metabolic end products (alanopine, strombine, lactate, acetate or propionate) were not detected. Succinate accumulation and net ATP breakdown were the fastest in the least resistant species, L. obtusata, and slowest in the most resistant, L. littorea. Rate of ATP turnover decreased during freshwater exposure in L. littorea and L. saxatilis, but not in L. obtusata. Our data suggest that differential resistance of three studied Littorina spp. to extreme hypoosmotic stress may be related to their different abilities to reduce metabolic rate and ATP turnover during sustained anoxia. Species-specific variations in anaerobic capacity of Littorina spp. are discussed in relation to their vertical distribution, size and ecology. Accepted: 4 October 1999     

The role of ion regulation in the control of the distribution of Gammarus tigrinus (Sexton) in salt-polluted rivers

Fluctuating salinities at different sites on the German salt-polluted rivers Werra and Weser were compared with extracellular ion levels of specimens of Gammarus tigrinus (Sexton; Amphipoda, Crustacea), collected at the same sites. G. tigrinus regulated haemolymph concentrations of inorganic anions (Cl⁻, SO²⁻ ₄, PO³⁻ ₄) and cations (Na⁺, K⁺, Mg²⁺, Ca²⁺) during fluctuations of salt pollution in the upper Weser. This capacity to regulate varying levels of salt pollution in the upper Weser, correlated well with the distribution of the brackish amphipods in this river ecosystem. G. tigrinus tolerated periods of Na⁺ and Cl⁻ stress (>380 mmol l⁻¹) without compensating these maxima by regulating extracellular Na⁺ and Cl⁻. However, during such bursts of Na⁺ and Cl⁻ stress in Werra and Weser, the ability to regulate extracellular [K⁺] at river water K⁺ stress of ≥6.0 mmol l⁻¹ may explain why this brackish species has been more successful in these rivers than its competitors like Gammarus pulex. The present investigation demonstrates that the water salinity affects the [NO⁻ ₃] in the haemolymph of G. tigrinus. With increasing hypo-osmotic stress the animals accumulate increasing amounts of NO⁻ ₃. A simultaneous increase in stream water [NO⁻ ₃] causes an additional accumulation of NO⁻ ₃ in the haemolymph. The high extent of accumulation indicates that active ion transport systems may be involved. The accumulation of NO⁻ ₃ in the haemolymph has low physiological consequences to G. tigrinus, but when hypo-osmotically stressed under anoxic conditions, nitrite formed by the reduction of nitrate may have an adverse affect on the metabolism of G. tigrinus. Accepted: 4 October 1999     

Ion channels in guard cells of Arabidopsis thaliana (L.) Heynh.

Despite the availability of many mutants for signal transduction, Arabidopsis thaliana guard cells have so far not been used in electrophysiological research. Problems with the isolation of epidermal strips and the small size of A. thaliana guard cells were often prohibiting. In the present study these difficulties were overcome and guard cells were impaled with double-barreled microelectrodes. Membrane-potential recordings were often stable for over half an hour and voltage-clamp measurements could be conducted. The guard cells were found to exhibit two states. The majority of the guard cells had depolarized membrane potentials, which were largely dependent on external K⁺ concentrations. Other cells displayed spontaneous transitions to a more hyperpolarized state, at which the free-running membrane potential (E_m) was not sensitive to the external K⁺ concentration. Two outward-rectifying conductances were identified in cells in the depolarized state. A slow outward-rectifying channel (s-ORC) had properties resembling the K⁺-selective ORC of Vicia faba guard cells (Blatt, 1988, J Membr Biol 102: 235–246). The activation and inactivation times and the activation potential, all depended on the reversal potential (E_rev) of the s-ORC conductance. The s-ORC was blocked by Ba²⁺ (K_1/2 = 0.3–1.3mM) and verapamil (K_1/2 = 15–20 μM). A second rapid outward-rectifying conductance (r-ORC) activated instantaneously upon stepping the voltage to positive values and was stimulated by Ba²⁺. Inward-rectifying channels (IRC) were only observed in cells in the hyperpolarized state. The activation time and activation potential of this channel were not sensitive to the external K⁺ concentration. The slow activation of the IRC (t_1/2 ≈ 0.5 s) and its negative activation potential (V_threshold = −155 mV) resemble the values found for the KAT1 channel expressed in Saccharomyces cerevisiae (Bertl et al., 1995, Proc Natl Acad Sci USA 92: 2701–2705). The results indicate that A. thaliana guard cells provide an excellent system for the study of signal transduction processes. Received: 28 March 1996 / Accepted: 11 November 1996     

Characterization of ATP-dependent proteolysis in embryos of the brine shrimp, Artemia franciscana

Under anoxia, embryos of Artemia franciscana enter a state of quiescence. During this time protein synthesis is depressed, and continued degradation of proteins could jeopardize the ability to recover from quiescence upon return to favorable conditions. In this study, we developed an assay for monitoring ATP/ubiquitin-dependent proteolysis in order to establish the presence of this degradation mechanism in A. franciscana embryos, and to describe some characteristics that may regulate its function during anoxia-induced quiescence. For lysates experimentally depleted of adenylates, supplementation with ATP and ubiquitin stimulated protein degradation rates by 92 ± 17% (mean ± SE) compared to control rates. The stimulation by ATP was maximal at concentrations ≥11 μmol · l⁻¹. In the presence of ATP and ubiquitin, ubiquitin-conjugated proteins were produced by lysates during the course of the 4-h assays, as detected by Western blotting. Acute acidification of lysates to values approximating the intracellular pH observed under anoxia completely inhibited ATP/ubiquitin-dependent proteolysis. Depressed degradation was also observed under conditions where net ATP hydrolysis occurred. These results suggest that ATP/ubiquitin-dependent proteolysis is markedly inhibited under cellular conditions promoted by anoxia. Inhibition of proteolysis during quiescence may be one critical factor that increases macromolecular stability, which may ultimately govern the duration of embryo survival under anoxia. Accepted: 2 November 1999     

Seizure-like afterdischarges simulated in a model neuron

To explore non-synaptic mechanisms in paroxysmal discharges, we used a computer model of a simplified hippocampal pyramidal cell, surrounded by interstitial space and a “glial-endothelial” buffer system. Ion channels for Na⁺, K⁺, Ca²⁺ and Cl⁻ _, ion antiport 3Na/Ca, and “active” ion pumps were represented in the neuron membrane. The glia had “leak” conductances and an ion pump. Fluxes, concentration changes and cell swelling were computed. The neuron was stimulated by injecting current. Afterdischarge (AD) followed stimulation if depolarization due to rising interstitial K⁺ concentration ([K⁺]_o) activated persistent Na⁺ current (I _Na,P). AD was either simple or self-regenerating; either regular (tonic) or burst-type (clonic); and always self-limiting. Self-regenerating AD required sufficient I _Na,P to ensure re-excitation. Burst firing depended on activation of dendritic Ca²⁺ currents and Ca-dependent K⁺ current. Varying glial buffer function influenced [K⁺]_o accumulation and afterdischarge duration. Variations in Na⁺ and K⁺ currents influenced the threshold and the duration of AD. The data show that high [K⁺]_o and intrinsic membrane currents can produce the feedback of self-regenerating afterdischarges without synaptic input. The simulated discharge resembles neuron behavior during paroxysmal firing in living brain tissue. Action Editor: David Terman     

Effects of cytoskeletal modifications on Ca2+ influx after cerebral ischemia

Summary.  The fungal toxin cytochalasin D as well as endogenous gelsolin depolymerize filamentous actin which may induce dynamic uncoupling of membrane ion channels. In vitro application of cytochalasin D reduced NMDA-induced [³H]noradrenaline release from mouse brain neocortical slices by 38%. In gsn deficient neocortical synaptosomes [Ca²⁺]_i increase in response to K⁺ (30 mM) depolarization was 33% higher than in wild-type. After transient focal cerebral ischemia K⁺-induced [Ca²⁺]_i increase in neocortical synaptosomes was 56% lower than in synaptosomes prepared from the non-ischemic contralateral hemisphere. After in vivo pretreatment with cytochalasin D 10 min before MCA occlusion K⁺-induced [Ca²⁺]_i increase in synaptosomes in vitro prepared 1 h after reperfusion from the ischemic hemisphere was only 25% lower than in contralateral synaptosomes, while cytochalasin D pretreatment in vivo did not reduce K⁺-induced [Ca²⁺]_i increase in vitro. Hence, presynaptic Ca²⁺ influx and subsequently neuronal vulnerability are attenuated by increased and are aggravated by decreased F-actin depolymerization. Received June 29, 2001 Accepted August 6, 2001 Published online August 9, 2002     

Ecophysiological characteristics of Avicennia germinans and Laguncularia racemosa coexisting in a scrub mangrove forest at the Indian River Lagoon, Florida

The purpose of this work is to increase ecological understanding of Avicennia germinans L. and Laguncularia racemosa (L.) Gaertn. F. growing in hypersaline habitats with a seasonal climate. The area has a dry season (DS) with low temperature and vapour pressure deficit (vpd), and a wet season (WS) with high temperature and slightly higher vpd. Seasonal patterns in interstitial soil water salinity suggested a lack of tidal flushing in this area to remove salt along the soil profile. The soil solution sodium/potassium (Na⁺/K⁺) ratio differed slightly along the soil profile during the DS, but during the WS it was significantly higher at the soil surface. Diurnal changes in xylem osmolality between predawn (higher) and midday (lower) were observed in both species. However, A. germinans had higher xylem osmolality compared to L. racemosa. Xylem Na⁺/K⁺ suggested higher selectivity of K⁺ over Na⁺ in both species and seasons. The water relations parameters derived from pressure–volume P–V curves were relatively stable between seasons for each species. The range of water potentials (Ψ), measured in the field, was within estimated values for turgor maintenance from P–V curves. Thus the leaves of both species were osmotically adapted to maintain continued water uptake in this hypersaline mangrove environment.     

Localization of ion-regulatory epithelia in embryos and hatchlings of two cephalopods

The tissue distribution and ontogeny of Na⁺/K⁺-ATPase has been examined as an indicator for ion-regulatory epithelia in whole animal sections of embryos and hatchlings of two cephalopod species: the squid Loligo vulgaris and the cuttlefish Sepia officinalis. This is the first report of the immunohistochemical localization of cephalopod Na⁺/K⁺-ATPase with the polyclonal antibody α (H-300) raised against the human α1-subunit of Na⁺/K⁺-ATPase. Na⁺/K⁺-ATPase immunoreactivity was observed in several tissues (gills, pancreatic appendages, nerves), exclusively located in baso-lateral membranes lining blood sinuses. Furthermore, large single cells in the gill of adult L. vulgaris specimens closely resembled Na⁺/K⁺-ATPase-rich cells described in fish. Immunohistochemical observations indicated that the amount and distribution of Na⁺/K⁺-ATPase in late cuttlefish embryos was similar to that found in juvenile and adult stages. The ion-regulatory epithelia (e.g., gills, excretory organs) of the squid embryos and paralarvae exhibited less differentiation than adults. Na⁺/K⁺-ATPase activities for whole animals were higher in hatchlings of S. officinalis (157.0 ± 32.4 μmol g_FM⁻¹ h⁻¹) than in those of L. vulgaris (31.8 ± 3.3 μmol g_FM⁻¹ h⁻¹). S. officinalis gills and pancreatic appendages achieved activities of 94.8 ± 18.5 and 421.8 ± 102.3 μmol_ATP g_FM⁻¹ h⁻¹, respectively. High concentrations of Na⁺/K⁺-ATPase in late cephalopod embryos might be important in coping with the challenging abiotic conditions (low pH, high pCO₂) that these organisms encounter inside their eggs. Our results also suggest a higher sensitivity of squid vs. cuttlefish embryos to environmental acid-base disturbances.     

Transport of Ca2+ across the tonoplast of intact vacuoles from Chenopodium album L. suspension cells: ATP-dependent import and inositol-1,4,5-trisphosphate-induced release

The removal of Ca²⁺ from the medium by intact vacuoles and microsomes of Chenopodium album was investigated by measuring INDO-1 fluorescence emission at 400 and 480 nm and the response of Ca²⁺ -selective mini-electrodes. The removal of Ca²⁺ depended on the presence of MgATP, displaying an apparent K _mATP of about 50 μM, a K _mCa of 400–500 nM, and a nucleotide specificity (%) of ATP (100) > CTP (49) > GTP (28) > UTP (20) > ADP = AMP (0). In the presence of saturating MgATP, the vacuoles reduced the [Ca²⁺] of the medium below 30 nM. Part of the Ca²⁺ removed from the medium was released again after adding micromolar concentrations of inositol-1,4,5-trisphosphate. This release of Ca²⁺ was inhibited by heparin. Since digitonin caused the release of the entire amount of Ca²⁺ removed from the medium in the presence of MgATP, we argue that the Ca²⁺ is not bound to membranes or sequestered otherwise, but is transported into the vacuoles (or vesicles) and remains freely mobile there. In accordance with the current literature, we conclude that the plant vacuole is an important store for mobile Ca²⁺ to be released for purposes of signal transduction. Since changes in the trans-tonoplast ΔpH and inhibition of the H⁺-translocating pumps had no significant influence on the ATP-dependent removal of Ca²⁺ from the cytoplasmic side, we argue that in C. album ATP-driven Ca²⁺ transport is the predominant form of Ca²⁺ translocation into the vacuole. Received: 11 July 1996 / Accepted: 18 October 1996     

Pathways for K<Superscript>+</Superscript> Efflux in Isolated Surface and Crypt Colonic Cells. Activation by Calcium

K⁺-conductive pathways were evaluated in isolated surface and crypt colonic cells, by measuring ⁸⁶Rb efflux. In crypt cells, basal K⁺ efflux (rate constant: 0.24 ± 0.044 min⁻¹, span: 24 ± 1.3%) was inhibited by 30 mM TEA and 5 mM Ba²⁺ in an additive way, suggesting the existence of two different conductive pathways. Basal efflux was insensitive to apamin, iberiotoxin, charybdotoxin and clotrimazole. Ionomycin (5 μM) stimulated K⁺ efflux, increasing the rate constant to 0.65 ± 0.007 min⁻¹ and the span to 83 ± 3.2%. Ionomycin-induced K⁺ efflux was inhibited by clotrimazole (IC₅₀ of 25 ± 0.4 μM) and charybdotoxin (IC₅₀ of 65 ± 5.0 nM) and was insensitive to TEA, Ba²⁺, apamin and iberiotoxin, suggesting that this conductive pathway is related to the Ca²⁺-activated intermediate-conductance K⁺ channels (IK_ca). Absence of extracellular Ca²⁺ did neither affect basal nor ionomycin-induced K⁺ efflux. However, intracellular Ca²⁺ depletion totally inhibited the ionomycin-induced K⁺ efflux, indicating that the activation of these K⁺ channels mainly depends on intracellular calcium liberation. K⁺ efflux was stimulated by intracellular Ca²⁺ with an EC₅₀ of 1.1 ± 0.04 μM. In surface cells, K⁺ efflux (rate constant: 0.17 ± 0.027 min⁻¹; span: 25 ± 3.4%) was insensitive to TEA and Ba²⁺. However, ionomycin induced K⁺ efflux with characteristics identical to that observed in crypt cells. In conclusion, both surface and crypt cells present IK_Ca channels but only crypt cells have TEA- and Ba²⁺-sensitive conductive pathways, which would determine their participation in colonic K⁺ secretion.     

Purinergic Modulation of Na+,K+,Cl− Cotransport and MAP Kinases is Limited to C11-MDCK Cells Resembling Intercalated Cells from Collecting Ducts

We demonstrated recently that in renal epithelial cells from collecting ducts of Madin-Darby canine kidneys (MDCK), Na⁺,K⁺,Cl⁻ cotransport is inhibited up to 50% by ATP via its interaction with P_2Y purinoceptors (Biochim. Biophys. Acta 1998. 1369:233–239). In the present study we examined which type of renal epithelial cells possesses the highest sensitivity of Na⁺,K⁺,Cl⁻ cotransport to purinergic regulation. We did not observe any effect of ATP on Na⁺,K⁺,Cl⁻ cotransport in renal epithelial cells from proximal and distal tubules, whereas in renal epithelial cells from rabbit and rat collecting ducts ATP decreased the carrier's activity by ∼30%. ATP did not affect Na⁺,K⁺,Cl⁻ cotransport in C7 subtype MDCK cells possessing the properties of principal cells but led to ∼85% inhibition of this carrier in C11-MDCK cells in which intercalated cells are highly abundant. Both C7- and C11-MDCK exhibited ATP-induced IP₃ and cAMP production and transient elevation of [Ca²⁺] _i . In contrast to the above-listed signaling systems, ATP-induced phosphorylation of ERK and JNK MAP kinases was observed in C11-MDCK only. Thus, our results reveal that regulation of renal Na⁺,K⁺,Cl⁻ cotransport by P_2Y receptors is limited to intercalated cells from collecting ducts and indicate the involvement of the MAP kinase cascade in purinergic control of this ion carrier's activity. Received: 10 June 1999/Revised: 23 August 1999     

Kinetics of K-Cl Cotransport in Frog Erythrocyte Membrane: Effect of External Sodium

In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO₃) has been shown to mediate a large fraction of the total K⁺ transport. In the present study, Cl⁻-dependent and Cl⁻-independent K⁺ fluxes via frog erythrocyte membranes were investigated as a function of external and internal K⁺ ([K⁺] _e and [K⁺] _i ) concentration. The dependence of ouabain-resistant Cl⁻-dependent K⁺ (⁸⁶Rb) influx on [K⁺] _e over the range 0–20 mm fitted the Michaelis-Menten equation, with an apparent affinity (K _m ) of 8.2 ± 1.3 mm and maximal velocity (V _max ) of 10.4 ± 1.6 mmol/l cells/hr under isotonic conditions. Hypotonic stimulation of the Cl⁻-dependent K⁺ influx increased both K _m (12.8 ± 1.7 mm, P < 0.05) and V _max (20.2 ± 2.9 mmol/l/hr, P < 0.001). Raising [K⁺] _e above 20 mm in isotonic media significantly reduced the Cl⁻-dependent K⁺ influx due to a reciprocal decrease of the external Na⁺ ([Na⁺] _e ) concentration below 50 mm. Replacing [Na⁺] _e by NMDG⁺ markedly decreased V _max (3.2 ± 0.7 mmol/l/hr, P < 0.001) and increased K _m (15.7 ± 2.1 mm, P < 0.03) of Cl⁻-dependent K⁺ influx. Moreover, NMDG⁺ Cl substitution for NaCl in isotonic and hypotonic media containing 10 mm RbCl significantly reduced both Rb⁺ uptake and K⁺ loss from red cells. Cell swelling did not affect the Na⁺-dependent changes in Rb⁺ uptake and K⁺ loss. In a nominally K⁺(Rb⁺)-free medium, net K⁺ loss was reduced after lowering [Na⁺] _e below 50 mm. These results indicate that over 50 mm [Na⁺] _e is required for complete activation of the K-Cl cotransporter. In nystatin-pretreated cells with various intracellular K⁺, Cl⁻-dependent K⁺ loss in K⁺-free media was a linear function of [K⁺] _i , with a rate constant of 0.11 ± 0.01 and 0.18 ± 0.008 hr⁻¹ (P < 0.001) in isotonic and hypotonic media, respectively. Thus K-Cl cotransport in frog erythrocytes exhibits a strong asymmetry with respect to transported K⁺ ions. The residual, ouabain-resistant K⁺ fluxes in NO₃ were only 5–10% of the total and were well fitted to linear regressions. The rate constants for the residual influxes were not different from those for K⁺ effluxes in isotonic (∼0.014 hr⁻¹) and hypotonic (∼0.022 hr⁻¹) media, but cell swelling resulted in a significant increase in the rate constants. Received: 19 November 1998/Revised: 23 August 1999     

Purinergic signalling in rat GFSHR-17 granulosa cells: an <Emphasis Type="Italic">in vitro</Emphasis> model of granulosa cells in maturing follicles

Purinergic signalling in rat GFSHR-17 granulosa cells was characterised by Ca²⁺-imaging and perforated patch-clamp. We observed a resting intracellular Ca²⁺-concentration ([Ca²⁺]_i) of 100 nM and a membrane potential of −40 mV. This was consistent with high K⁺− and Cl⁻ permeability and a high intracellular Cl⁻ concentration of 40 mM. Application of ATP for 5–15 s every 3 min induced repeated [Ca²⁺]_i increases and a 30 mV hyperpolarization. The phospholipase C inhibitor U73122 or the IP₃-receptor antagonist 2-aminoethoethyl diphenyl borate suppressed ATP responses. Further biochemical and pharmacological experiments revealed that ATP responses were related to stimulation of P2Y₂ and P2Y₄ receptors and that the [Ca²⁺]_i increase was a prerequisite for hyperpolarization. Inhibitors of Ca²⁺-activated channels or K⁺ channels did not affect the ATP-evoked responses. Conversely, inhibitors of Cl⁻ channels hyperpolarized cells to −70 mV and suppressed further ATP-evoked hyperpolarization. We propose that P2Y₂ and P2Y₄ receptors in granulosa cells modulate Cl⁻ permeability by regulating Ca²⁺-release.     

ATP-dependent potassium channels of muscle cells: Their properties,regulation, and possible functions

ATP-dependent potassium channels are present at high density in the membranes of heart, skeletal, and smooth muscle and have a lowP _open at physiological [ATP]_i. The unitary conductance is 15–20 pS at physiological [K⁺] _o , and the channels are highly selective for K⁺. Certain sulfonylureas are specific blockers, and some K channel openers may also act through these channels. K_ATP channels are probably regulated through the binding of ATP, which may in turn be regulated through changes in the ADP/ATP ratio or in pH_i. There is some evidence for control through G-proteins. The channels have complex kinetics, with multiple open and closed states. The main effect of ATP is to increase occupancy of long-lived closed states. The channels may have a role in the control of excitability and probably act as a route for K⁺ loss from muscle during activity. In arterial smooth muscle they may act as targets for vasodilators.     

Characterization of a K+-ATPase from Lactobacillus helveticus ATCC 15009

Lactobacillus helveticus ATCC 15009 (wild-type) membrane preparations hydrolyzed Mg²⁺-ATP as a function of K⁺ concentration (2–200 mM). Mg²⁺-ATP hydrolysis by L. helveticus membranes was strongly inhibited in the absence of exogenous K⁺, while it amounted to 6 nmol ATP hydrolyzed min^–1 (mg membrane protein)^–1 at 50 mM KCl (saturating conditions) and pH 7.2. The K⁺-dependent ATPase of L. helveticus displayed a relatively high affinity for potassium ions (K _m = 800 μM) and was not affected by pretreatment of membranes with N,N’-dicyclohexylcarbodiimide. Membrane preparations were subjected to hypotonic shock to obtain a maximum yield of open profiles. The formation of a maximum level of enzyme-phosphate complex with a molecular mass of approximately 82 kDa was induced upon treatment of L. helveticus membrane preparations with low concentrations of [γ-³²P]ATP in the presence of K⁺ and La³⁺ ions and was visualized by acidic SDS-PAGE. It was concluded that L. helveticus membranes contain an inwardly directed K⁺ pump whose presence is discussed in terms of its putative role in cytoplasmic pH regulation. Received: 16 December 1996 / Accepted: 14 May 1997     

Membrane responses to changes in the extracellular potassium concentration in isolated hippocampal pyramidal neurons

The responses of freshly isolated hippocampal pyramidal neurons to rapid, elevations of the external potassium concentration ([K⁺] _out ) were investigated using the whole-cell variation of a patch-clamp technique. An elevation of [K⁺] _out induced a two-phase inward current at the membrane potentials more negative than the reversal potential for K ions. This current consisted of a leakage, current and a time-dependent current (τ=40–50 msec at 21°C), the latter designated below asI _ΔK. It displayed first-order activation kinetics that showed neither voltage, nor concentration dependence. The amplitude of this current was determined by the external K⁺ concentration and increased with hyperpolarization. Voltage dependence ofI _ΔK measured within the range from −20 to −120 mV was similar to that for inward rectifier. Activation ofI _ΔK was utterly dependent on Na⁺; substitution of extracellular Na⁺ with choline chloride almost completely depressedI _ΔK.I _ΔK was absent in the cells freshly dissociated from the nodosal and dorsal root ganglia. This suggests that this earlier unrecognized current is instrumental in preserving densely packed hippocampal pyramidal neurons from sudden increases in [K⁺] _out and following spontaneous over-excitation. It prevents the neurons from responding to K⁺-induced depolarizations by slowing down potassium influx.