The cardioplegic solution HTK: effects on membrane potential,intracellular K+ and Na+ activities in sheep cardiac Purkinje fibres

The effects of the cardioplegic solution HTK on membrane potential (E_M) and intracellular K and Na activities (a _K ⁱ , a _Na ⁱ ) were studied in sheep cardiac Purkinje fibres by means of conventional and ion-selective microelectrodes. HTK contains (mM): Na 15, K 10, Ca 0, Mg 4, histidine 180, (1) In control conditions E_M was –74.3±3.3 mV (n=25), a _K ⁱ was 116.4±4.1 mM (n=7) and a _Na ⁱ was 8.2±1.4 mM (n=15). (2) Exposure to HTK led to a depolarization to –59.7±3.6 mV (n=25) which exceeded by about 5–7 mV that induced in a Tyrode solution of 10 mM K and in a modified HTK solution supplemented by 2 mM Ca (n=6). (3) Addition of 0.5 mM barium eliminated the difference in the steady-state depolarization. (4) HTK superfusion increased a _K ⁱ to 120.1±4.4 mM (n=7) and decreased a _Na ⁱ to 3.9±0.9 mM (n=15). (5) The decrease in a _Na ⁱ was insensitive to amiloride (1 mM) and to external alkalization but was slightly increased by addition of 2 mM calcium. (6) When the calcium in Tyrode solution was lowered from 2.0 mM to 0.05 mM, a _Na ⁱ hardly decreased during subsequent exposure to unmodified HTK and it increased in the presence of 0.1 mM dihydroouabain. We propose the hypothesis (1) that the difference in membrane depolarization between HTK and a 10 mM K-Tyrode is caused by a decrease in K conductance by the HTK solution and (2) that the a _Na ⁱ decline mainly results from a coupled Ca influx via Na-Ca exchange due to a delayed washout of external calcium.This work was supported by the Deutsche Forschungsgemeinschaft, SFB 330 — Organprotektion     

Cell Na+ activities and transcellular Na+ absorption by descending colon from normal and Na+-deprived rabbits

The relation between intracellular Na⁺ activities, (Na)_c, determined employing Na⁺-selective microelectrodes, and the rates of active Na⁺ absorption,I _Na, by rabbit descending colon was examined whenI _Na was varied over a wide range by chronic dietary Na⁺ deprivation. (Na)_c averaged 13 mM and was independent ofI _Na over a sixfold range. Further, the ratios of the slope resistance of the apical membrane (r ^m) to that of the basolateral membrane (r ^s) (i.e.r ^m/r ^s) in low-transporters (control diet) and high-transporters (Na⁺-deprived) did not differ significantly inspite of the fact that the Na⁺ conductance of the apical membranes of high-transporters was, on the average, three times greater than that of the low-transporters. These findings, together with the results reported by other laboratories, strongly suggest that the aldosterone-induced increase in the conductance of the apical membrane to Na⁺ and, in turn, the rate of entry of Na⁺ into the absorptive cells are followed by parallel increases in the ability of cells to extrude Na⁺ across the basolateral membrane in the absence of a sustained increase in (Na)_c as well as the conductance of that barrier.     

Na+ and pH dependence of proline and beta-alanine absorption in rat small intestine

Aims: Early characterization of intestinal absorption of imino acids in mammals has demonstrated the existence of a Na⁺‐dependent, Cl^?‐independent transport system in rat small intestine, which is the only carrier for β‐alanine. Based on the substrate selectivity, it was proposed that the Proton Amino Acid Transporter 1 (PAT1) could be the same as this imino acid carrier. The present study characterizes the pH and Na⁺ dependence of proline and β‐alanine uptake in rat small intestine. Methods: Intestinal uptake of radiolabelled l ‐proline or β‐alanine was measured in brush border membrane vesicles and everted intestinal rings, in the presence and absence of Na⁺ and at different pH values. Results: The existence of an inwardly directed H⁺ gradient in the absence of Na⁺ enhanced the initial entry of proline and β‐alanine in brush border membrane vesicles, that reached a transient overshoot with maximal value around 30 s. In the absence of pH gradient, no overshoot was shown. In entire tissue, there was an increase of proline and β‐alanine uptake at acidic pH that was higher in the presence of Na⁺ than in its absence. This ion dependence and pH effect of the amino acids uptake also increased with the incubation period. Substrate inhibition studies confirmed that intestinal proline absorption in rat occurs mainly by system B and PAT1‐like transporter. Conclusions: There is a Na⁺‐independent, H⁺‐dependent transporter of amino acids at the apical membrane of the rat enterocytes.     

Intrinsic difference in erythrocyte membrane in spontaneously hypertensive rats characterized by Na+ and K+ fluxes

The goad of this study was to determine whether the elevated flux of sodium and potassium through the erythrocyte membrane of spontaneously hypertensive rats (SHR) is due to an intrinsic difference in the cell membrane or to a humoral factor present in the plasma. Isolated and washed erythrocytes from SHR and normotensive Wistar Kyoto (WKy) and Sprague-Dawley (SD) rats, were incubated in 1) a physiological salt solution, 2) WKy or SD plasma and 3) SHR plasma. Incubations were performed at 4°C for 23 h. Erythrocytes from SHR incubated in physiological salt solution had significantly greater Na⁺ and K⁺ fluxes than those from normotensive WKy and SD rats (P<0.005). Plasma from any of the three strains of rats, as compared to physiological salt solution, increased Na⁺ influx in the following order: SD>WKy>SHR. Erythrocyte K⁺ efflux was not altered by plasma. We conclude that the elevated flux of Na⁺ and K⁺ in SHR erythrocytes is due to an intrinsic difference in the cell membrane. The greater Na⁺ influx in plasma from any strain of rats is not correlated with the blood pressure of the rat. The lesser increase in Na⁺ influx in crythrocytes incubated in plasma from SHR masks the greater intrinsic membrane permeability in the SHR erythrocyte when Na⁺ fluxes are studied in whole blood. The elevated flux of Na⁺ and K⁺ through the erythrocyte membrane of SHR may reflect a general membrane defect that underlies the pathogenesis of elevated arterial pressure.     

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

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

Role of Na+ and K+ in enzyme function

Metal complexation is a key mediator or modifier of enzyme structure and function. In addition to divalent and polyvalent metals, group IA metals Na+ and K+ play important and specific roles that assist function of biological macromolecules. We examine the diversity of monovalent cation (M+)-activated enzymes by first comparing coordination in small molecules followed by a discussion of theoretical and practical aspects. Select examples of enzymes that utilize M+ as a cofactor (type I) or allosteric effector (type II) illustrate the structural basis of activation by Na+ and K+, along with unexpected connections with ion transporters. Kinetic expressions are derived for the analysis of type I and type II activation. In conclusion, we address evolutionary implications of Na+ binding in the trypsin-like proteases of vertebrate blood coagulation. From this analysis, M+ complexation has the potential to be an efficient regulator of enzyme catalysis and stability and offers novel strategies for protein engineering to improve enzyme function.     

K+-Stimulated Na+ transport in frog-skin epithelia

Increasing [K⁺] from 2.5 mmol/l to 115 mmol/l on the serosal side of the frog skin produces a rapid decrease of short-circuit current (I_sc) that is followed, within a few minutes, by a recovery of I_sc to near or above its control value. After isolation of the epithelium by a procedure involving collagenase treatment and physical removal of thecorium, increasing serosal [K⁺] still produced a depression of I_sc but no significant recovery phase. By itself, collagenase treatment reduced but did not eliminate the recovery phase. The recovery phase was also markedly depressed by the beta-adrenergic blocker oxprenolol, but not by propranolol, atropine or indomethacin. Amiloride, given during the recovery phase, caused I_sc to reverse to a small outward value. These results suggest that the recovery phase of I_sc seen in the response to increased serosal [K⁺] represents an increase in Na⁺ influx through amiloride-sensitive channels which is triggered by the release of an intermediary agent, possibly a beta-adrenergic agonist, from some structure in thecorium.     

Molecular cloning and functional characterization of the Aplysia FMRFamide-gated Na+ channel

FMRFamide-gated Na⁺ channel (FaNaC) is the only known peptide-gated ion channel, which belongs to the epithelial Na⁺ channel/degenerin (ENaC/DEG) family. We have cloned a putative FaNaC from the Aplysia kurodai CNS library using PCR, and examined its characteristics in Xenopus oocytes. A. kurodai FaNaC (AkFaNaC) comprised with 653 amino acids, and the sequence predicts two putative membrane domains and a large extracellular domain as in other members of the ENaC/DEG family. In oocytes expressing AkFaNaC, FMRFamide evoked amiloride-sensitive Na⁺ current. Different from the known FaNaCs (Helix and Helisoma FaNaCs), AkFaNaC was blocked by external Ca²⁺ but not by Mg²⁺. Also, desensitization of the current was enhanced by Mg²⁺ but not by Ca²⁺. The FMRFamide-gated current was depressed in both low and high pH. These results indicate that AkFaNaC is an FaNaC of Aplysia, and that the channel has Aplysia specific functional domains.     

The Na+-activated K+ channel contributes to K+ efflux in Na+-loaded guinea-pig but not rat ventricular myocytes

Activation of the Na+-activated K+ channels (KNa channels) has been suggested to contribute to the ischaemia-induced accumulation of extracellular K+ (K+e) in the mammalian myocardium. Recent evidence shows that these channels are not present in rat ventricular myocytes [9]. We have therefore investigated the effect of raised intracellular Na+ activity (aiNa) on intracellular K+ activity (aiK) in guinea-pig myocytes, which possess the channels, and on rat ventricular myocytes which do not. The Na+-activated K+ current was activated by an increase in aiNa induced by removing extracellular Ca2+ and Mg2+ and inhibiting the Na-pump. The aiNa increased and the aiK decreased in both guinea-pig and rat myocytes superfused with Ca2+- and Mg2+-free Tyrode. The new steady-state increase in aiNa and decline in aiK were similar in both species. Inhibition of the Na-pump resulted in an additional increase in aiNa and decrease in aiK in both species. However, both the increase in aiNa and decrease in aiK were greater in guinea-pig myocytes and the decline in aiK in guinea-pig myocytes followed the development of a large Na+-activated K+ current. When Li+ replaced Na+ in the superfusate the Na+-activated K+ current did not develop and the fall in aiK was reduced. In Na+-loaded rat myocytes, which do not have a Na+-activated K+ current, the decline in aiK was reduced and blocked by 2 mM Mg2+ suggesting that a Mg2+-sensitive non-specific cation channel may be involved in the K+ efflux from rat myocytes [12]. These data suggest that KNa channels are a major route for K+ efflux from Na+-loaded guinea-pig myocytes.     

Na^＋，K^＋—ATPase研究进展

Na+ ,K+ ATPase广泛分布于多种细胞的细胞膜上 ,是维持细胞内外Na+ ,K+ 浓度梯度的关键酶。Na+ ,K+ ATPase由α,β和γ亚基组成 ,在不同组织 ,不同发育阶段表达不同的亚型。激素等通过蛋白激酶A、蛋白激酶C、酪氨酸激酶等调节Na+ ,K+ ATPase的活性。小的跨膜蛋白结合Na+ ,K+ ATPase的特异亚基 ,调节其活性。Na+ ,K+ ATPase不仅参与离子转运、蛋白转运、维持离子自稳平衡 ,而且在脊椎动物胚胎发育、神经元导向、中枢神经系统发育、细胞形态维持、细胞粘附等方面发挥作用 ,甚至可作为喹巴因的受体 ,参与信号传递。     

Na+ and K+ concentration of rat parotid saliva

The effects of carbachol and auriculo-temporal stimulation on the Na⁺ and K⁺ concentrations of rat parotid saliva have been compared. The main duct perfused in situ, does not transport Na⁺ or K⁺ and is water impermeable. The Na⁺ concentration of secretion evoked by either stimulus is flow dependent, increasing with increasing flow rate and plateauing at near plasma Na⁺ levels. At low flow rate the carbachol evoked secretion has a higher Na⁺ concentration. This is not due to the release of catecholamines since neither sympathectomy nor adrenoceptor block altered the nature of the secretion. The K⁺ concentration, whilst flow dependent, decreasing with increasing flow rate, was the same for both stimuli.     

High extracellular K+ evokes changes in voltage-dependent K+ and Na+ currents and volume regulation in astrocytes

[K⁺]_e increase accompanies many pathological states in the CNS and evokes changes in astrocyte morphology and glial fibrillary acidic protein expression, leading to astrogliosis. Changes in the electrophysiological properties and volume regulation of astrocytes during the early stages of astrocytic activation were studied using the patch-clamp technique in spinal cords from 10-day-old rats after incubation in 50 mM K⁺. In complex astrocytes, incubation in high K⁺ caused depolarization, an input resistance increase, a decrease in membrane capacitance, and an increase in the current densities (CDs) of voltage-dependent K⁺ and Na⁺ currents. In passive astrocytes, the reversal potential shifted to more positive values and CDs decreased. No changes were observed in astrocyte precursors. Under hypotonic stress, astrocytes in spinal cords pre-exposed to high K⁺ revealed a decreased K⁺ accumulation around the cell membrane after a depolarizing prepulse, suggesting altered volume regulation. 3D confocal morphometry and the direct visualization of astrocytes in enhanced green fluorescent protein/glial fibrillary acidic protein mice showed a smaller degree of cell swelling in spinal cords pre-exposed to high K⁺ compared to controls. We conclude that exposure to high K⁺, an early event leading to astrogliosis, caused not only morphological changes in astrocytes but also changes in their membrane properties and cell volume regulation.     

Angiotensin II desensitization and Ca2+ and Na+ fluxes in vascular smooth muscle cells

The role of ion fluxes in angiotensin II (AII) desensitization (tachyphylaxis) was investigated by studying Na⁺ and Ca²⁺ translocation in cultured vascular smooth muscle cells from the rat aorta. The effects of AII were compared to those of [1-sarcosine]-AII (Sar¹-AII), an analogue which also induces tachyphylaxis, and [2-lysine]-AII (Lys²-AII), an analogue that does not show this property. Maximally effective concentrations of the three peptides induced a rapid and transient increase in ⁴⁵Ca²⁺ efflux, a rapid and sustained decrease in total cell Ca²⁺ and an increased Na⁺ permeability. Repeated treatments, at short intervals, with either of the three peptides abolished the effect on Ca²⁺ efflux, and this desensitization was slowly reversible. A 30-min rest period was sufficient for full recovery of the response of cells that were desensitized by Lys²AII, whereas the recovery from AII or Sar¹AII-desensitization was still not complete after 60 min. Our results suggest that the difference in the behaviour of the tachyphylactic AII and Sar¹-AII and the non-tachyphylactic Lys²-AII lays not in the production of different signals upon binding to the receptor, but in a difference in the hormone-receptor interaction itself.     

Na~+/K~+-ATP酶与糖尿病血管并发症

综述Na+ /K+- ATP酶在糖尿病血管并发症的活性和表达变化 ,以揭示糖尿病发病过程中有多种因素参与该酶的调节 ,造成血管的通透性、收缩血液流变学改变 ,参与血管并发症发生。     

Decreased expression of apical Na+ channels and basolateral Na+, K+-ATPase in ulcerative colitis

Impaired absorption of sodium (Na+) and water is a major factor in the pathogenesis of diarrhoea in ulcerative colitis (UC). Electrogenic Na+ absorption, present mainly in human distal colon and rectum, is defective in UC, but the molecular basis for this is unclear. The effect of UC on the expression of apical Na+ channels (ENaC) and basolateral Na+, K+-ATPase, the critical determinants of electrogenic Na+ transport, was therefore investigated in this study. Sigmoid colonic and/or proximal rectal mucosal biopsies were obtained from patients with mild to moderate UC, and patients with functional abdominal pain (controls). ENaC subunit expression was studied by immunohistochemistry, western blot analysis, and in situ hybridization, and Na+, K+-ATPase isoform expression was studied by immunohistochemistry, western blotting, and northern blot analysis. UC was associated with substantial decreases in the expression of the ENaC beta- and gamma-subunit proteins and mRNAs, whereas the decrease in ENaC alpha-subunit protein detected by immunolocalization was less marked. The levels of expression of Na+, K+-ATPase alpha1- and beta1-isoform proteins were also lower in UC patients than in controls, although there were no differences in Na+, K+-ATPase alpha1- and beta1-isoform mRNA levels between the two groups. Taken together, these results show that UC results mainly in decreased expression of the apical ENaC beta- and gamma-subunits, as well as the basolateral Na+, K+-ATPase alpha1- and beta1-isoforms. In conclusion, these changes provide a basis for the low/negligible levels of electrogenic Na+ absorption seen in the distal colon and rectum of UC patients, which contribute to the pathogenesis of diarrhoea in this disease.     

Choroid plexus, brain and kidney Na+,K+-ATPase: comparative activities in fetal, newborn and young adult rabbits

We have studied maturation of brain barrier systems in fetal, newborn, juvenile and adult rabbits. We have compared choroid plexus and brain Na+,K+-ATPase levels in each age group, as well as serum and CSF Na+ concentration as a measure of the ability of the choroid plexus to generate a gradient from blood to CSF. The choroid plexus appears functionally mature at all ages studied, both in ability to produce a Na+ gradient and in ATPase levels. In contrast, brain ATPase levels rose markedly with age. Kidney ATPase measured for comparison showed a pattern similar to brain.     

Modulation of intracellular Na+ and Ca2+ induced by the cardiac Na+-Ca2+ exchanger in guinea pig ventricular myocytes

The Na+-Ca2+ exchanger current was measured in single guinea pig ventricular myocytes, using the whole-cell voltage-clamp technique, and intracellular free calcium concentration ([Ca2+](i)) was monitored simultaneously with the fluorescent probe Indo-1 applied intracellularly through a perfused patch pipette. In external solutions, which have levels of Ca2+ (approximately 66 microM Ca2+) thought low enough to inhibit exchanger turnover, the removal of external Na+ (by replacement with Li+) induced both an outward shift of the holding current and an increase in [Ca2+](i), even though the recording pipette contained 30 mM bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), sufficient to completely block phasic contractions. The effects of Na+ removal were blocked either by the extracellular application of 2 mM Ni2+ or by chelating extracellular Ca2+ with 1 mM EGTA. In the presence of 10 microM Ryanodine, the effects of external Na+ substitution with Li(+) on both membrane current and [Ca2+](i) were attenuated markedly in amplitude and at a much slower time course. Reversal potentials were estimated by using ramp pulses and by defining exchange currents as the Ni2+-sensitive components. The experimental values of the reversal potential and [Ca2+](i) were used to calculate cytosolic Na+ ([Na+](i)) by assuming an exchanger stoichiometry of 3Na+ : 1Ca2+. These calculations suggested that in the nominal absence of external Ca2+ ( approximately 66 microM under our experimental conditions), the exchanger operates at -40 mV as though approximately 40 mM Na+ had accumulated in the vicinity of the intracellular binding sites. We conclude that under the conditions of low extracellular Ca2+ and high intracellular Ca2+ buffering, the Na+-Ca2+ exchanger can still generate sufficient Ca2+ influx on the removal of external Na+ to markedly increase cytosolic free Ca2+.     

Effects of ATP and elevated K+ on K+ currents and intracellular Ca2+ in human microglia.

We have used whole-cell patch-clamp recordings and calcium microfluorescence measurements to study the effects of ATP and elevated external K+ on properties of human microglia. The application of ATP (at 0.1 mM) led to the activation of a transient inward non-selective cationic current at a cell holding potential of -60 mV and a delayed, transient expression of an outward K+ current activated with depolarizing steps applied from holding level. The ATP response included an increase in inward K+ conductance and a depolarizing shift in reversal potential as determined using a voltage ramp waveform applied from -120 to -50 mV. Fura-2 microspectrofluorescence measurements showed intracellular calcium to be increased following the application of ATP. This response was characterized by an initial transient phase, which persisted in Ca2+-free media and was due to release of Ca2+ from intracellular storage sites. The response had a later plateau phase, consistent with Ca2+ influx. In addition, ATP-induced changes in intracellular Ca2+ exhibited prominent desensitization. Elevated external K+ (at 40 mM) increased inward K+ conductance and shifted the reversal potential in the depolarizing direction, with no effect on outward K+ current or the level of internal Ca2+. The results of these experiments show the differential responses of human microglia to ATP and elevated K+, two putative factors associated with neuronal damage in the central nervous system.