Activation of PKC increases Na+-K+ pump current in ventricular myocytes from guinea pig heart

 We have previously shown activation of α₁-adrenergic receptors increases Na⁺-K⁺ pump current (I _p) in guinea pig ventricular myocytes, and the increase is eliminated by blockers of phosphokinase C (PKC). In this study we examined the effect of activators of PKC on I _p. Phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased I _P at each test potential without shifting its voltage dependence. The concentration required for a half-maximal response (K _0.5) was 6 μM at 15 nM cytosolic [Ca²⁺] ([Ca²⁺]_i) and13 nM at 314 nM [Ca²⁺]_i. The maximal increase at either [Ca²⁺]_i was about 30%. Another activator of PKC, 1,2-dioctanoyl-sn-glycerol (diC₈), increased I _p similarly. The effect of PMA on I _P was eliminated by the PKC inhibitor staurosporine, but not by the peptide PKI, an inhibitor of protein kinase A (PKA). PMA and α₁-adrenergic agonist effects both were sensitive to [Ca²⁺]_i, blocked by PKC inhibitors, unaffected by PKA inhibition, and increased I _p uniformly at all voltages. However, they differed in that α₁-activation caused a maximum increase of 15% vs 30% via PMA, and α₁-effects were less sensitive to [Ca²⁺]_i than PMA effects. These results demonstrate that activation of PKC causes an increase in I _p in guinea pig ventricular myocytes. Moreover, they suggest that the coupling of α₁-adrenergic activation to I _p is entirely through PKC, however α₁-activation may be coupled to a specific population of PKC whereas PMA is a more global agonist. Received: 21 August 1998 / Received after revision: 7 December 1998 / Accepted: 11 December 1998     

Ion gradients and contractility in skeletal muscle: the role of active Na+, K+ transport

Intensive contractile activity is associated with a significant net loss of K⁺ and a comparable gain of Na⁺ in the working muscle fibres. This leads to an increase in the interstitial and T-tubular K⁺ concentration and to a decrease in the T-tubular Na⁺ concentration. It is well established that the exposure of muscles to high extracellular K⁺ or low extracellular Na⁺ inhibits contractile performance. More importantly, the combination of high extracellular K⁺ and low extracellular Na⁺ has a much more pronounced inhibitory effect on force than the sum of the individual effects of the two ions. The inhibitory effects of high extracellular K⁺ or low extracellular Na⁺ can be alleviated within 5–10 min by acute hormonal stimulation of the Na⁺, K⁺ pump. In contrast, reductions in the capacity for active Na⁺, K⁺ transport by pre-incubation of isolated muscles with ouabain or by prior K⁺ depletion of the animals significantly decreases contractile endurance during high-frequency electrical stimulation. Thus, muscles from K⁺-depleted rats exhibiting a 54% reduction in Na⁺, K⁺ pump concentration showed a 110% increase in force decline during 30 s of 60 Hz stimulation. Reducing the Na⁺, K⁺ pump capacity to a similar extent by pre-incubation with ouabain led to a comparable decrease in endurance. Moreover, reductions in the Na⁺, K⁺ pump capacity were associated with an increased intracellular accumulation of Na⁺ during electrical stimulation. These observations support the notion that excitation-induced decreases in Na⁺, K⁺ gradients contribute to fatigue during intensive exercise and suggest that the capacity for active Na⁺, K⁺ transport is a determining factor for contractile endurance.     

The significance of active Na+,K+ transport in the maintenance of contractility in rat skeletal muscle

The effects of reduced Na⁺,K⁺ pump capacity on contractile endurance and excitation-induced changes in intracellular Na⁺ content were investigated in isolated rat soleus and extensor digitorum longus muscles. Pre-incubation with 10^-5m ouabain increased the rate of force decline measured over the first 5–20 s of tetanic contraction from 0.32 to 0.94% s^-1 and 1.4 to 4.6% s^-1 in soleus and extensor digitorum longus muscles, respectively. Soleus muscles from K⁺-deficient rats exhibited 54% reduction in the concentration of Na⁺,K⁺ pumps and the force decline during 30 s of 60 Hz stimulation was increased from 0.53 to 1.15% s^-1. A similar change was induced in control muscles when a comparable reduction in the concentration of functional Na⁺,K⁺ pumps was elicited by pre-incubation with ouabain (10^-6-2×10^-6m ). In soleus, the force decline during 60 s of 60 Hz stimulation showed linear correlation to the increase in intracellular Na⁺ content. In extensor digitorum longus, force decline and increase in Na⁺ content during 60 Hz stimulation were both four times faster than in soleus as measured over 15 s of excitation. These results indicate that during maximal contractions the Na⁺,K⁺ pump capacity is one of the determinants for the contractile endurance in skeletal muscle. Furthermore, the maintenance of contractile force seems to be a function of the rate of Na⁺-influx and this relationship may account for the difference in endurance between slow-twitch and fast-twitch muscles.     

人肝再生增强因子在体外可与Na+-K+-ATPase 特异性结合

目的： 用体外下拉实验证明人肝再生增强因子（hALR）与其相互作用蛋白Na+-K+-ATPase的体外结合作用。方法：将Na+-K+-ATPase β亚基部分基因片段定向克隆到pGEX-4T-1 中, 转化大肠杆菌DH5α，IPTG 诱导, 获得Na+-K+-ATPase β亚基部分蛋白与谷胱甘肽（GST）的融合表达，经GST偶联的琼脂糖珠纯化, 以GST下拉实验检测其与hALR蛋白的体外直接相互作用。结果：还原型SDS-PAGE 显示GST- Na+-K+-ATPase 融合蛋白泳道有hALR蛋白单体和二聚体条带，Western blotting 结果进一步证实为hALR蛋白。结论：Na+-K+-ATPase可在体外与hALR蛋白特异地结合。     

Intracellular Na+ measurements in smooth muscle using SBFI – changes in [Na+], Ca2+ and force in normal and Na+-loaded ureter

 Our understanding of the control and effects of intracellular [Na⁺] ([Na⁺]_i) in intact smooth muscle is limited by the lack of data concerning [Na⁺]_i. The initial aim of this work was therefore to investigate the suitability of using the Na⁺-sensitive fluorophore SBFI in intact smooth muscle. We find this to be a good method for measuring [Na⁺]_i in ureteric smooth muscle. Resting [Na⁺]_i was found to be around 10 mM and rose to 25 mM when the Na⁺-K⁺-ATPase was inhibited by ouabain. This relatively low [Na⁺]_i in the absence of Na⁺-K⁺-ATPase suggests that other cellular processes, such as Na⁺-Ca²⁺ exchange, play a role in maintaining [Na⁺]_i under these conditions. Simultaneous measurements of [Na⁺]_i or [Ca²⁺] _i and force showed that Na⁺-Ca²⁺ exchange can play a functional role in ureteric smooth muscle. We found that the greater the driving force for Na⁺ exit and hence Ca²⁺ entry, the larger the contraction. In addition the Na⁺-Ca²⁺ exchanger activity under these conditions was found to be pH sensitive: acidification reduced the contraction and concomitant changes in [Ca²⁺] and [Na⁺]_i. We conclude that SBFI is a useful method for monitoring [Na] in smooth muscle and that Na⁺-Ca²⁺ exchange may play a functional role in the ureter. Received: 26 August 1997 / Received after revision: 27 October 1997 / Accepted: 28 October 1997     

Cation pumps in skeletal muscle: potential role in muscle fatigue.

Two membrane bound pumps in skeletal muscle, the sarcolemma Na⁺-K⁺ adenosine triphosphatase (ATPase) and the sarcoplasmic reticulum Ca²⁺-ATPase, provide for the maintenance of transmembrane ionic gradients necessary for excitation and activation of the myofibrillar apparatus. The rate at which the pumps are capable of establishing ionic homeostasis depends on the maximal activity of the enzyme and the potential of the metabolic pathways for supplying adenosine triphosphate (ATP). The activity of the Ca²⁺-ATPase appears to be expressed in a fibre type specific manner with both the amount of the enzyme and the isoform type related to the speed of contraction. In contrast, only minimal differences exist between slow-twitch and fast-twitch fibres in Na⁺-K⁺ ATPase activity. Evidence is accumulating that both active transport of Na⁺ and K⁺ across the sarcolemma and Ca²⁺-uptake by the sarcoplasmic reticulum may be impaired in vivo in a task specific manner resulting in loss of contractile function. In contrast to the Ca²⁺-ATPase, the Na⁺-K⁺ ATPase can be rapidly upregulated soon after the onset of a sustained pattern of activity. Similar programmes of activity result in a downregulation of Ca²⁺-ATPase but at a much later time point. The manner in which the metabolic pathways reorganize following chronic activity to meet the changes in ATP demand by the cation pumps and the degree to which these adaptations are compartmentalized is uncertain.     

Effect of strenuous strength training on the Na-K pump concentration in skeletal muscle of well-trained men

This study examined how strenuous strength training affected the Na-K pump concentration in the knee extensor muscle of well-trained men and whether leg muscle strength and endurance was related to the pump concentration. First, the pump concentration, taken as ³H-ouabain binding, was measured in top alpine skiers since strength training is important to them. Second, well-trained subjects carried out strenuous eccentric resistance training either 1, 2, or 3 times · week⁻¹ for 3 months. The Na-K pump concentration, the maximal muscle strength in a full squat lift (one repetition maximum, 1 RM), and the muscle endurance, taken as the number of full squat lifts of a mass of 70% of the 1 RM load, were measured before and after the training period. The mean pump concentration of the alpine skiers was 425 (SEM 11) nmol · kg⁻¹ wet muscle mass. The subjects in part two increased their maximal strength in a dose-dependent manner. The muscle endurance increased for all subjects but independently of the training programme. From a mean starting value of 356 (SEM 6) nmol · kg⁻¹ the mean Na-K pump concentration increased by 54 (SEM 15) nmol · kg⁻¹ (+15%, P < 0.001) when the results for all subjects were pooled. The effect was larger for those who had trained twice a week than for those who had trained only once a week (P=0.025), suggesting that the effect of strength training depended on the amount of training carried out. The muscle strength and endurance were not related to the pump concentration, suggesting that the pumping power of this enzyme did not limit the performance during heavy lifting. However, the individual improvements in the endurance test during the training period correlated with the individual changes in the pump concentration (r _Spearman=0.5; P=0.01) which could mean that a common factor both increases the pump concentration and makes the muscles more adapted to repeated heavy lifting. Accepted: 8 August 2000     

Amiloride-inhibitable Na+ conductance in rat proximal tubule

 Previous single-channel recordings from the luminal membrane of the rabbit proximal tubule have revealed amiloride-inhibitable Na⁺ channels of a characteristic conductance range. The present study aimed to pursue this issue in rat proximal tubule. Control rats were compared to those put on a low-Na⁺ diet or pretreated by triamcinolone injections (s.c.). Stimulation of Na⁺ absorption by glucocorticoids was verified by examining the transepithelial voltage in Ussing chamber studies of the distal colon. The membrane voltage (V _m) of isolated, in-vitro-perfused proximal tubule segments was measured in patch-clamp and impalement studies. It was found that amiloride hyperpolarized V _m significantly by 2.1 ± 0.9 mV (n = 26) in tubules of control rats, by 3.9 ± 0.7 mV (n = 12) in rats put on a low-Na⁺ diet and by 3.7 ± 1.0 mV (n = 17) in rats treated with glucocorticoids. The effect of amiloride was concentration dependent with a half-maximal effect at < 1 μmol/l. RT-PCR techniques were used to search for the presence of the α-, β- and γ-subunits of the epithelial Na⁺ channel in isolated oximal tubule segments. The presence of the respective mRNAs was verified. These data indicate that: (1) amiloride-inhibitable Na⁺ channels are present in rat proximal tubules; (2) the Na⁺ conductance may be upregulated by Na⁺ deprivation but is still very limited when compared to total cell conductance; (3) therefore, the contribution of Na⁺-channel-mediated absorption to total proximal Na⁺ absorption is probably small. Received: 5 August 1996 / Received after revision: 22 January 1997 / Accepted: 28 January 1997     

The regulation of the Na+,K+ pump in contracting skeletal muscle

Increased passive Na⁺,K⁺ fluxes necessitate an efficient activation of the Na⁺,K⁺ pump in working muscles to limit the rundown of the Na⁺,K⁺ chemical gradients and ensuing loss of excitability. Several studies have demonstrated an increase in Na⁺,K⁺-pump rate in working muscles, and in electrically stimulated muscles up to a 22-fold increase in active Na⁺,K⁺ transport has been observed. Excitation-induced increase in intracellular Na⁺ is believed to be the primary stimulus for Na⁺,K⁺ pumping in a contracting muscle. In muscles recovering from electrical stimulation, however, the activity of the pump may stay elevated even after intracellular Na⁺ has been reduced to below the resting level. Moreover, in rat soleus muscles 10-s stimulation at 60 Hz induced a 5-fold increase in the activity of the Na⁺,K⁺ pump although mean intracellular [Na⁺] was unchanged. These findings strongly suggest that a substantial part of the excitation-induced increase in Na⁺,K⁺-pump activity is caused by mechanisms other than increased intracellular [Na⁺]. The mechanism behind this activation is not clear, but may involve a change in the affinity of the Na⁺,K⁺ pump for intracellular Na⁺. In addition to intracellular [Na⁺], the Na⁺,K⁺ pump may be stimulated in contracting muscles by other factors such as catecholamines, calcitonin gene-related peptide (CGRP), free fatty acids and cytoskeletal links. Together, this activation may form a feed forward mechanism protecting muscles from loss of excitability during periods of contraction by increasing Na⁺,K⁺-pump activity prior to erosion of the Na⁺,K⁺ chemical gradients. During exercise of high intensity, however, intracellular [Na⁺] increases substantially constituting an additional stimulus for the pump.     

Changes in K+, Na+ and calcium contents during in vivo stimulation of rat skeletal muscle

The effects of in vivo stimulation via the sciatic nerve on Na⁺ K⁺ and calcium contents in slow-twitch and fast-twitch muscles were compared. Whereas intermittent stimulation for 24 h at 20 Hz caused only minor changes in soleus (SOL), a considerable loss of K⁺ (around 24%) and gain of Na⁺ (around 84%) was observed in extensor digitorum longus (EDL) and tibialis anterior (TA) muscles. These changes could be detected within 0.5 h and a plateau was maintained from 2 to 24 h. Total calcium content increased progressively, reaching values 245 and 382% above the control level in EDL and TA muscle, respectively, after 24 h of 20 Hz stimulation. Whereas the Na⁺ and K⁺ content recovered within a few hours, calcium content did not return towards control level until after 48 h of rest. In a pilot study performed with continuous stimulation at 10 Hz, the changes in Na⁺ and K⁺ contents in SOL, EDL and TA muscle were comparable to those at 20 Hz. The concentration of the Na⁺-K⁺ pumps was highest in the fast-twitch EDL and TA muscles and was unaffected by 10 Hz stimulation. It is concluded that a stimulation pattern leading to a rise in intracellular Na⁺ and a loss of K⁺ may cause a marked accumulation of calcium. These events seem to be related to insufficient activation of the Na-K⁺ pump rather than to variations in the total Na⁺-K' pump capacity.     

Effect of dehydration on apical Na+-H+ exchange activity and Na+-dependent sugar transport in brush-border membrane vesicles isolated from chick intestine

 The current work examines the effect of 4 days of water deprivation on Na⁺-H⁺ exchange and Na⁺-sugar cotransport systems in brush-border membrane vesicles isolated from either the jejunum, ileum or the colon of the chick. Apical Na⁺-H⁺ exchange activity was evaluated by measuring the pH-gradient-dependent Na⁺ uptake. The contribution of the Na⁺-H⁺ exchangers NHE2 and NHE3 to total Na⁺-H⁺ exchange activity was evaluated from their sensitivity to the amiloride-related drug HOE694. Dehydration increased plasma aldosterone levels from 12 to 70 pg/ml and also the activities of both Na⁺-H⁺ exchange and Na⁺-dependent sugar transport in the three intestinal regions tested. Na⁺-independent sugar transport was not modified by 4 days of water deprivation. In the ileum and colon the increase in Na⁺-H⁺ exchange activity was due to an increase in NHE2 activity, whereas in the jejunum it was due to an increase in both NHE2 and NHE3. Since we have previously reported that chronic Na⁺ depletion increases plasma aldosterone levels and NHE2 activity in ileum and colon, decreased small and large intestine Na⁺-sugar cotransport activity and had no effect on jejunal apical Na⁺-H⁺ exchange activity, it can be concluded that: (1) aldosterone does not regulate intestinal Na⁺-dependent sugar transport, and (2) the regulation of jejunal Na⁺-H⁺ exchange activity differs from that of either the ileum or the colon. Received: 31 October 1997 / Received after revision: 17 December 1997 / Accepted: 8 January 1998     

Inhibition of the Na+,K+ pump by the epileptogenic pentylenetetrazole

 Pentylenetetrazole (PTZ) is a convulsant drug used in animal experiments to induce epileptic activity. It is known to interact with a variety of channels and neurotransmitter receptors. We investigated the effects of PTZ on the Na⁺,K⁺ pump by measuring pump-mediated current, ⁸⁶Rb⁺ uptake and [³H]ouabain binding using the Xenopus oocytes as a model system. PTZ inhibits Rb⁺ uptake with a K _I value of about 10 mM, and the number of pump molecules is not altered as judged by the number of ouabain-binding sites. The measurements of pump current under voltage-clamp conditions revealed voltage-dependent inhibition with K _I values increasing with depolarization. Reduced sensitivity to PTZ in the presence of ouabain and at reduced external K⁺ suggests that PTZ directly interacts with the transport ATPase and binds preferentially to the E1 form. Development of inhibition and recovery have time constants of about 10 min, which suggests that the externally applied PTZ acts on the pump from the cytoplasmic or membrane phase. Since inhibition of the Na⁺,K⁺ pump is accompanied by membrane depolarization, PTZ should promote the generation of discharges and contribute to the epileptogenic effects through pump inhibition. Received: 28 May 1998 / Received after revision: 7 August 1998 / Accepted: 20 August 1998     

Effects of thyroid hormones on contractility and cation transport in skeletal muscle

Skeletal muscle is one of the major target organs for thyroid hormone. The muscles most commonly affected are those used during prolonged effort (slow-twitch muscles). One of the major clinical features is the shortening of the Achilles-tendon reflex time in hyperthyroidism and its prolongation in hypothyroidism. Most of the peripheral effects of the thyroid hormones can be ascribed to the action of triiodothyronine (T₃), which is produced by de-iodination of thyroxine (T₄) in liver and kidney. From the plasma, T₃ is actively transported into skeletal muscle. The Ca²⁺ ATPase in skeletal muscle is responsible for removal of Ca²⁺ ions from the cytosol into the sarcoplasmic reticulum (SR) during relaxation, and the Na⁺, K⁺ ATPase in the plasma membrane is responsible for restoration of the membrane potential after excitation. The concentrations of Ca²⁺ ATPase and Na⁺, K⁺ ATPase in rat skeletal muscle have been shown to increase four- and 10-fold, respectively, in the transition from the hypothyroid to the hyperthyroid state. In humans, a linear correlation between the Na⁺, K⁺ ATPase concentration of skeletal muscle and the free T₄ index was established. Significant effects of T₃ on Ca²⁺ ATPase and Na⁺, K⁺ ATPase can be detected 24 h after a single injection. These effects are mediated by increased production of mRNA for the respective proteins, initiated by binding of T₃ to nuclear receptors. Passive fluxes of Ca²⁺, Na⁺ and K⁺ also show a significant rise after T₃ treatment. The increase in passive fluxes of Na⁺ and K⁺ can be detected before the rise in the concentration of Na⁺, K⁺ ATPase, suggesting that T₃, in addition to its nuclear effects, may have a direct effect on the plasma membrane. Apart from their significance for muscle function in thyroid disease, the changes in Ca²⁺ ATPase and Na⁺, K⁺ ATPase will be important in other conditions where T₃ and T₄ levels show dramatic changes, i.e. during postnatal development, starvation and undernutrition, as well as in non-thyroidal illness (low-T₃ syndrome).     

Effect of chloride on Ca2+ release from the sarcoplasmic reticulum of mechanically skinned skeletal muscle fibres

 The effect of intracellular Cl^– on Ca²⁺ release in mechanically skinned fibres of rat extensor digitorum longus (EDL) and toad iliofibularis muscles was examined under physiological conditions of myoplasmic [Mg²⁺] and [ATP] and sarcoplasmic reticulum (SR) Ca²⁺ loading. Both in rat and toad fibres, the presence of 20 mM Cl^–in the myoplasm increased Ca²⁺ leakage from the SR at pCa (i.e. –log₁₀ [Ca²⁺]) 6.7, but not at pCa 8. Ca²⁺ uptake was not significantly affected by the presence of Cl^–. This Ca²⁺-dependent effect of Cl^– on Ca²⁺ leakage was most likely due to a direct action on the ryanodine receptor/Ca²⁺ release channel, and could influence channel sensitivity and the resting [Ca²⁺] in muscle fibres in vivo. In contrast to this effect, acute addition of 20 mM Cl^– to the myoplasm caused a 40–50% reduction in Ca²⁺ release in response to a low caffeine concentration both in toad and rat fibres. One possible explanation for this latter effect is that the addition of Cl^– induces a potential across the SR (lumen negative) which might reduce Ca²⁺ release via several different mechanisms. Received: 20 October 1997 / Received after revision: 1 December 1997 / Accepted: 2 December 1997     

窒息新生儿脐血D-二聚体水平和红细胞膜Na~+-K~+-ATP酶活性变化

目的:分析窒息新生儿脐血pH、D-二聚体水平及红细胞膜Na+-K+-ATP酶活性变化。方法 :选择临产过程出现急性胎儿窘迫孕妇40例,其剖宫娩出新生儿以1min Apgar评分确定为正常者20例(窘迫组),出现窒息者20例(窒息组);另选无急性胎儿窘迫、同样剖宫娩出的正常新生儿20例作为对照组。取各组脐动脉血,血气分析仪检测pH值,免疫比浊法检测D-二聚体水平,定磷法检测红细胞膜Na+-K+-ATP酶活性。比较各组上述指标的差异。结果:方差分析显示,各组脐动脉血pH值、D-二聚体水平和红细胞膜Na+-K+-ATP活性差异均有统计学意义(P0.05)。窒息组脐动脉血pH值明显低于对照组和窘迫组(P均0.05),D-二聚体水平显著高于对照组和窘迫组(P均0.05),红细胞膜Na+-K+-ATP酶活性显著低于对照组与窘迫组(P均0.05);窘迫组pH值和红细胞膜Na+-K+-ATP酶活性显著低于对照组(P均0.05)。结论 :窒息新生儿纤溶和红细胞膜泵功能检测结果,可为新生儿窒息治疗措施的选择提供实验依据。     

Fuzzy space and control of Na+, K+-pump rate in heart and skeletal muscle

Since intracellular Na⁺ activity (aⁱ_Na) is one important determinant of Na⁺, K⁺-pump rate as well as excitability and the finely tuned contractility, it is surprising that the relation between aⁱ_Na and pump rate reported by different authors has k_0.5 varying between 10 and 40 mmol L^-1. Other data also point to a variable relation between pump rate and aⁱ_Na. During stimulation of isolated rat soleus muscles at 2 Hz, ouabain-sensitive ⁸⁶Rb uptake was increased in spite of the intracellular Na⁺ remaining unaltered. In isolated cardiomyocytes, a transient Na⁺, K⁺-pump current was observed upon activation by extracellular K⁺ in spite of good control of aⁱ_Na. Na⁺-loaded, isolated, sheep cardiac Purkinje fibres initially hyperpolarized over a period of up to 1 min upon activation of the Na⁺, K⁺ pump with no detectable change of aⁱ_Na. These examples are compatible with the existence of a micro-environment close to the membrane where diffusion is slower than in the rest of the cytosol, so that local aⁱ_Na may fluctuate or gradients may develop as visualized by Wendt-Gallitelli et al. (1993). We conclude that the reported relationships between Na⁺, K⁺-pump rate and aⁱ_Na in intact cells probably underestimate the true affinity of the Na⁺, K⁺ pump for Na⁺ due to a functional diffusion barrier beneath the sarcolemma, and also because of incomplete cell dialysis in whole-cell voltage clamp experiments. The Na⁺, K⁺ pump seems to be preferentially supplied with Na⁺ from the outside through neighbouring channels and transporters.     

A Na+-activated K+ current (I K,Na) is present in guinea-pig but not rat ventricular myocytes

 The effects of removing extracellular Ca²⁺ and Mg²⁺ on the membrane potential, membrane current and intracellular Na⁺ activity (a ⁱ _Na) were investigated in guinea-pig and rat ventricular myocytes. Membrane potential was recorded with a patch pipette and whole-cell membrane currents using a single-electrode voltage clamp. Both guinea-pig and rat cells depolarize when the bathing Ca²⁺ and Mg²⁺ are removed and the steady-state a ⁱ _Na increases rapidly from a resting value of 6.4± 0.6 mM to 33±3.8 mM in guinea-pig (n=9) and from 8.9±0.8 mM to 29.3±3.0 mM (n=5) in rat ventricular myocytes. Guinea-pig myocytes partially repolarized when, in addition to removal of the bathing Ca²⁺ and Mg²⁺, K⁺ was also removed, however rat cells remained depolarized. A large diltiazem-sensitive inward current was recorded in guinea-pig and rat myocytes, voltage-clamped at –20 mV, when the bathing divalent cations were removed. When the bathing K⁺ was removed after Ca²⁺ and Mg²⁺ depletion, a large outward K⁺ current developed in guinea-pig, but not in rat myocytes. This current had a reversal potential of –80±0.7 mV and was not inhibited by high Mg²⁺ or glybenclamide indicating that it is not due to activation of non-selective cation or adenosine triphosphate (ATP)-sensitive K channels. The current was not activated when Li⁺ replaced the bathing Na⁺ and was blocked by R-56865, suggesting that it was due to the activation of K_Na channels. Received: 15 October 1998 / Received after revision: 22 January 1999 / Accepted: 5 February 1999     

Vanadate stimulates the pumped movements of Na in skeletal muscle

We have measured the effects of concentrations of vanadate ranging between 0.01 and 10 mM on the²²Na efflux of frog sartorius muscles. The addition of vanadate had no effects when concentrations lower than 0.5 mM were used; higher concentrations increased Na efflux. The increase was abolished by the addition of ouabain (10^–5M). In muscles pretreated with ouabain vanadate did not modify Na efflux. The stimulatory effects of vanadate on Na efflux were also observed in Na-free solutions indicating that the effux of vanadate was not caused mainly either by an increase in the exchange of Na for Na or by an increase in Na entry into the muscle. We also examined the effects of vanadate on muscles immersed in solutions containing 20 mM K⁺; both vanadate and increased K⁺ produced stimulations of Na efflux that were additive. Similarly when the effects of vanadate and insulin were measured on the Na efflux of the same muscle, additive effects were found. As the ouabain-sensitive Na efflux in frog muscle is generally agreed to be due to the activity of the Na-K ATPase, our findings suggest that the net effect of vanadate in intact muscle cells is an increase in the activity of the Na pump. Since vanadate affects many enzymes it is quite possible that the stimulatory action is not due to a direct effect on the Na-K ATPase but may be mediated through an intermediary step. Regardless of the specific mechanism, it is evident that, our results as well as other findings in the literature, strongly indicate that Na pumping by intact cells can be increased by vanadate administration. Hence it is not justified to attribute the physiological modifications caused by vanadate administration to blockade of the Na-K ATPase unless the attribution is justified by specific experimental evidence.