A newly identified Ca2+ dependent K+ channel in the smooth muscle membrane of single cells dispersed from the rabbit portal vein

We found a new type of Ca²⁺-dependent K⁺ channel in smooth muscle cell membranes of single cells of the rabbit portal vein. A slope conductance of the current was 180 pS when 142 mM K⁺ solution was exposed to both sides of the membrane (this channel was named the K_M channel, in comparison to the known K_L and K_S channels from the same membrane patch; Inoue et al. 1985). This K_M channel was less sensitive to the cytoplasmic Ca²⁺ concentration, [Ca²⁺]_i, but was sensitive to the extracellular Ca²⁺, [Ca²⁺]_o, e.g. in the outside-out membrane patch, lowering the [Ca²⁺]_o in the bath markedly reduced the open probability of this channel, and also in cell-attached configuration, lowering of the [Ca²⁺]_o using the internally perfused patch clamp electrode device reduced the opening of K_M channel. TEA⁺ (1–10 mM) reduced the amplitude of the elementary current through the K_M channel applied from each side of the membrane, but this agent inhibited the K_M channel to a greater extent when applied to the inner than to the outer surface of the membrane. Furthermore, this K_M channel had a weak voltage dependency, and the open probability of the channel remained much the same within a wide range of potential (from –60 mV to +60 mV). Whereas most Ca²⁺-dependent K⁺ channels are regulated mainly by [Ca²⁺]_i and possess a voltage dependency, these properties of the K_M channel differed from other Ca²⁺-dependent K⁺ channels. The elucidation of this K_M channel should facilitate explanations of the actions of external Ca²⁺ or TEA⁺ on the membrane potential, in the smooth muscles of the rabbit portal vein.     

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.     

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.     

Evidence for a modulating effect of NA+/H+ exchange on the metabolic response of rat brown adipose tissue

Membrane potential and intracellular pH (pHi) were simultaneously monitored in rat perifused brown adipose tissue fragments by means of double-barrelled microelectrodes. In parallel experiments, the respiratory rate was measured. The cytosolic pH of unstimulated brown adipocytes was about 0.5 units higher than the value expected for a passive transmembrane distribution of H ions. Isoproterenol (5·10⁻¹⁰ M) had no effect on pHi and membrane potential while it induced a 3.1±0.5-fold increase of the respiratory rate. Clonidine (10⁻⁷ M) alone was followed by a cytosolic alkalinization of 0.14±0.05 pH units with no concomitant increase in the respiratory rate. A mirror image of the intracellular alkalinization induced by clonidine, i.e. an acidification of 0.09±0.03, was noted by monitoring the extracellular pH. Addition of clonidine in the presence of isoproterenol induced an alkalinization of 0.17±0.03 pH units and a 7.7±1.0-fold increase of the respiratory rate. Thus the alkalinizing effect of clonidine developed despite a massive increase in CO₂ production. Pretreatment of the preparation with amiloride (10⁻³ M), an inhibitor of Na⁺/H⁺ exchange, completely prevented the alkalinization and markedly reduced the potentiating effect of clonidine on the isoproterenol-induced respiratory rate. The results obtained are compatible with the hypothesis of a modulating effect of Na⁺/H⁺ exchange on the brown adipocyte metabolic response to catecholamine stimulation.     

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.     

Ketamine blocks voltage-gated K+ channels and causes membrane depolarization in rat mesenteric artery myocytes

Clinical doses of ketamine typically increase blood pressure, heart rate, and cardiac output. However, the precise mechanism by which ketamine produces these cardiovascular effects remains unclear. The voltage-gated K⁺ (K_V) channel is the major regulator of resting membrane potential (E _m) and vascular tone in many arteries. Therefore, we sought to evaluate the effects of ketamine on K_V currents using the standard whole-cell patch clamp recordings in single myocytes, enzymatically dispersed from rat mesenteric arteries. Ketamine [(±)-racemic mixture] inhibited K_V currents reversibly and concentration dependently with a K _d of 566.7 ± 32.3 μM and Hill coefficient of 0.75 ± 0.03. The inhibition of K_V currents by ketamine was voltage independent, and the time courses of channel activation and inactivation were little affected. The effects of ketamine on steady-state activation and inactivation curves were also minimal. Use-dependent inhibition was not observed either. S(+)-ketamine inhibited K_V currents with similar potency and efficacy as the racemic mixture. The average resting E _m in rat mesenteric artery myocytes was −44.1 ± 4.2 mV, and both racemic and S(+)-ketamine induced depolarization of E _m (15.8 ± 3.6 and 24.3 ± 5.0 mV at 100 μM, respectively). We conclude that ketamine induces E _m depolarization in vascular myocytes by blocking K_V channels in a state-independent manner, which may contribute to the increased vascular tone and blood pressure produced by this drug under a clinical setting.     

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.     

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     

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.     

Down-regulated Na+/K+-ATPase activity in ischemic penumbra after focal cerebral ischemia/reperfusion in rats

This study was aimed to examine whether the Na⁺/K⁺ adenosine triphosphatase (Na⁺/K⁺-ATPase) activity in ischemic penumbra is associated with the pathogenesis of ischemia/reperfusion-induced brain injury. An experimental model of cerebral ischemia/reperfusion was made by transient middle cerebral artery occlusion (tMCAO) in rats and the changes of Na⁺/K⁺-ATPase activity in the ischemic penumbra was examined by Enzyme Assay Kit. Extensive infarction was observed in the frontal and parietal cortical and subcortical areas at 6 h, 24 h, 48 h, 3 d and 7 d after tMCAO. Enzyme Assay analyses revealed the activity of Na⁺/K⁺-ATPase was decreased in the ischemic penumbra of model rats after focal cerebral ischemia/reperfusion compared with sham-operated rats, and reduced to its minimum at 48 h, while the infarct volume was enlarged gradually. In addition, accompanied by increased brain water content, apoptosis-related bcl-2 and Bax proteins, apoptotic index and neurologic deficits Longa scores, but fluctuated the ratio of bcl-2/Bax. Correlation analysis showed that the infarct volume, apoptotic index, neurologic deficits Longa scores and brain water content were negatively related with Na⁺/K⁺-ATPase activity, while the ratio of bcl-2/Bax was positively related with Na⁺/K⁺-ATPase activity. Our results suggest that down-regulated Na⁺/K⁺-ATPase activity in ischemic penumbra might be involved in the pathogenesis of cerebral ischemia/reperfusion injury presumably through the imbalance ratio of bcl-2/Bax and neuronal apoptosis, and identify novel target for neuroprotective therapeutic intervention in cerebral ischemic disease.     

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.     

Evidence for contribution of Ca2+ storage sites on unitary K+ channel currents in inside-out membrane of rabbit portal vein

While making use of the inside-out membrane patch, we examined the effects of caffeine and heparin on unitary currents of the large conductance Ca²⁺ -dependent K⁺ (maxi-K⁺) channel in the rabbit portal vein. About half of the inside-out membranes we used contained a functional Ca²⁺ -store site which facilitated modification of the maxi-K⁺ channel.When high-K⁺ solution containing 0.05mM EGTA was superfused in the bath, simultaneous openings of more than 20 maxi-K⁺ channels were observed in 39 of 83 patch membranes, and multi-channel opening appeared periodically or continuously at the holding potential of – 10mV. Most channel activities of these patch membranes were inhibited by caffeine or heparin, and some heparin-insensitive channel activities were inhibited by caffeine. The remaining patch membranes (44 out of 83) showed low activity of the maxi-K⁺ channel, and neither caffeine nor heparin modified channel activity.Therefore, in our experimental set-up, half the number of excised patch membranes contained a Ca²⁺ store site. Most Ca²⁺ store sites have inositol 1,4,5-trisphosphate (InsP₃)-activated Ca²⁺ release (IACR) and caffeine-activated Ca²⁺ release (CACR) channels and few lack the IACR channel. The mechanisms of activation of the maxi-K⁺ channel in relation to release of Ca²⁺ from the store sites can be examined in detail using the approaches we have described.     

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.     

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.     

Influence of Mitragyna Ciliata (MYTA) on the Microsomal Activity of ATPase Na+/K+ Dependent Extract on a Rabbit Heart

Mitragyna ciliata (MYTA) (Rubiaceae) inhibits plasmodia activity. MYTA induces a cardiotonicity of the digitalic type on rat''s isolated heart. In this work we studied the effect of MYTA on microsomal Na⁺/K⁺ dependant ATPase (Na⁺, K⁺ ATPase) extracted from the heart of a rabbit since digitalics inhibit Na⁺, K⁺ ATPase. Our results revealed that the Na⁺/K⁺ ATPase has an optimum pH of 7.4 and temperature of 37°C respectively. There is a linear relationship between the organic phosphate formed and the incubation time over 25 mins incubation period. The ATP hydrolysis rate in the presence of MYTA was 0.775 µM/min. LINEWEAVER and BURK plots showed that MYTA did not alter K_M (1.31 mM) but decreased V_MAX. This study shows that MYTA exerts a non-competitive inhibition on the microsomal Na⁺/K⁺ ATPase extracted from rabbit heart with a Ci₅₀ of 48 µg / ml. We conclude that the mechanism of action of MYTA is linked to the inhibition of the Na⁺/K⁺ ATPase like cardiotonics of the digitalic type.     

ATP-Dependent inhibition of Ca2+-activated K+ channels in vascular smooth muscle cells by neuropeptide Y

Neuropeptide Y(NPY) inhibits Ca²⁺-activated K⁺ channels reversibly in vascular smooth muscle cells from the rat tail artery. NPY (200 M) had no effect in the absence of intracellular adenosine 5triphosphate (ATP) and when the metabolic poison cyanide-M-chlorophenyl hydrozone (10 M) was included in the intracellular pipette solution. NPY was also not effective when ATP was substituted by the non-hydrolysable ATP analogue adenosine 5-[, -methylene]-triphosphate (AMP-PCP). NPY inhibited Ca²⁺-activated K⁺ channel activity when ATP was replaced by adenosine 5-O-(3-thiotriphosphate) (ATP [-S]) and the inhibition was not readily reversed upon washing. Protein kinase inhibitor (1 M), a specific inhibitor of adenosine 3, 5-cyclic monophosphatedependent protein kinase, had no significant effect on the inhibitory action of NPY. The effect of NPY on single-channel activity was inhibited by the tyrosine kinase inhibitor genistein (10 M) but not by daidzein, an inactive analogue of genistein. These observations suggest that the inhibition by NPY of Ca²⁺-activated K⁺ channels is mediated by ATP-dependent phosphorylation. The inhibitory effect of NPY was antagonized by the tyrosine kinase inhibitor genistein.     

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.     

Ouabain-induced isoform-specific localization change of the Na+, K+-ATPase alpha subunit in the synaptic plasma membrane of rat brain

Na+, K+-ATPase is one of major membrane proteins that has two subunits, alpha and beta. The alpha subunit has the ATPase activity and the ouabain binding site. Among four isoforms of the alpha subunit, expression of alpha1, alpha2, and alpha3, but not alpha4, is observed in matured rat brain. Ouabain is one of cardiac glycosides, and endogenous ouabain-like compounds have been recognized as a new class of steroid hormone. The alpha subunit is considered as their endogenous receptor. Recent studies envisaged the importance of membrane microdomains (MDs) as signaling platforms, which are recovered as a detergent-resistant membrane microdomain fraction (DRM). Although this ATPase has been considered as a non-DRM protein, some amount of the alpha subunit was found to be a component of the DRM prepared from the synaptic plasma membrane fraction (SPM) of rat brain. Ouabain treatment increased the amount of alpha3 isoform, but not alpha1, in the DRM derived from synaptosome fraction and SPM. These results suggest that the localization of the alpha subunit of Na+, K+-ATPase is regulated with isoform-specific mechanisms and the physiological importance of DRM in the signal transduction of the endogenous ouabain-like steroid hormone in neurons.     

Effects of urea on K+ fluxes and cell volume in perfused rat liver

Exposure of the perfused rat liver to a perfusate made hyperosmotic by the presence of 200 mmol l^–1glucose led, as expected, to marked, transient hepatocellular shrinkage followed by volume-regulatory net K⁺ uptake. However, even after this volume-regulatory K⁺ uptake had ceased, the liver cells are still slightly shrunken. Withdrawal of glucose from the perfusate resulted in marked transient cell swelling, net K⁺ release from the liver and restoration of cell volume. However, when the Krebs-Henseleit perfusate was made hyperosmotic by the presence of urea (20–300 mM), there was no immediate decrease in liver mass, yet a slight and persistent cell shrinkage developing 2 min after the onset of exposure to urea. Surprisingly, urea induced concentration-dependent net K⁺ efflux from the liver and removal of urea net K⁺ reuptake from the inflowing perfusate. The urea (200 mM)-induced net K⁺ release resembled that observed following a lowering of the influent [NaCl]: making the perfusate hypoosmotic (245 mosmol l^–1, by reducing influent [NaCl] by 30 mM) gave roughly the same K⁺ response as hyperosmotic exposure (505 mosmol/l) resulting from the presence of 200 mM urea. The urea-induced K⁺ efflux was not inhibited in the presence of ouabain (1 mM), or in Ca⁺⁺-free perfusion, but was modified in the presence of quinidine (1 mM) or Ba⁺⁺ (1 mM). The direction in which the liver was perfused had no effect on the urea-induced net K⁺ release. Electrophysiological studies showed that urea led to quinidine-sensitive hyperpolarization and increase in K⁺ selectivity of plasma membranes, suggesting opening of K⁺ channels in the hepatocyte plasma membrane in response to urea. The data suggest that urea, but not glucose, enters the hepatocyte as quickly as water. Furthermore, urea at high concentrations apparently leads to K⁺ efflux from the hepatocyte and cell shrinkage, possibly due to opening of K⁺ channels in the hepatocyte plasma membrane.