Studies on sarcolemma components may be misleading due to inadequate recovery.
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Animals↗
Biomedical subjects
T Clausen
Publications and source records attributed to T Clausen.
Thrombin-inhibitor complexes in the blood during and after delivery.
Activation of coagulation leads to generation of thrombin which in turn is inactivated by the formation of thrombin-antithrombin (TAT) complexes, and thrombin-heparin cofactor complexes (T-HCII). These complexes were measured in plasma by ELISA methods. During normal delivery, the median TAT level in ten women increased from 4.1 to 7.8 times the median normal reference level. There was great individual variation, and levels 42 and 56 times normal median were found in two women shortly after normal delivery. The median T-HCII levels increased only moderately from 2.3 to 3.1 times median normal reference. D-dimer values were elevated in 28 out of the 30 samples. In blood sampled 1-2 days after delivery, the median TAT level was 2.5 times the median normal reference. The median T-HCII level was now 5.6 times the median normal reference value. The values were stable during the first 4 days post partum, and there was little difference between those delivered vaginally or by Caesarean section (C-section). D-dimer values were above normal reference in all women, and higher in women delivered by C-section. In conclusion, increasing TAT levels during labour and delivery indicated generation of thrombin which was mainly inactivated by antithrombin. The T-HCII levels increased less during delivery. In the early post partum period, the T-HCII levels were relatively more increased than the TAT levels. These results suggest that intravascularly generated thrombin is preferably inactivated by antithrombin, even in parturient women. In the post partum period, formation of T-HCII complexes was more evident, possibly reflecting extravascular inactivation of thrombin.
Adult↗
Cloning, purification, and crystallization of Escherichia coli cystathionine beta-lyase.
The metC gene coding for cystathionine beta-lyase of Escherichia coli has been cloned and used to construct an overproducing E. coli strain. An efficient purification scheme has been developed and the purified enzyme has been crystallized by the hanging drop vapour diffusion method using either ammonium sulfate or polyethyleneglycol 400 as precipitating agent. The crystals belong to the orthorombic space group C222. Their unit cell parameters are a = 60.9 A, b = 154.7 A and c = 152.7 A. Consideration of the possible values of VM accounts for the presence of one dimer per asymmetric unit. The crystals are suitable for X-ray analysis and a complete native date set to 1.83 A resolution has been collected using synchrotron radiation.
Base Sequence↗
The Na+, K+ pump in skeletal muscle: quantification, regulation and functional significance.
In skeletal muscle, the Na+, K+ pump is predominantly situated in the sarcolemma (1000-3500 pumps per microns 2). The total concentration can be determined in fresh or frozen biopsies (1-5 mg) using a 3H-ouabain binding assay. The values obtained have been confirmed by measurements of maximum ouabain suppressible Na+, K(+)-transport capacity in intact muscles as well as Na+, K(+)-ATPase-related enzyme activity in muscle homogenates. In the mature organism, the concentration of Na+, K+ pumps varies with muscle type and species in the range 150-600 pmol (g wet wt)-1 in rat and human muscle, the concentration increases markedly with thyroid status. Semi-starvation and untreated diabetes reduce the concentration by 20-48%. K+ deficiency leads to a downregulation of up to 75%. Both in animals and in humans, training increases the concentration of Na+, K+ pumps in muscle and inactivity leads to a downregulation. High-frequency stimulation elicits up to a 20-fold increase in the net efflux of Na+ within 10 s This is the major activation mechanism for the Na+, K+ pump, utilizing its entire capacity and possibly represents a drive on de novo synthesis of Na+, K+ pumps. A variety of hormones (insulin, insulin-like growth factor I, adrenaline, noradrenaline, calcitonin gene-related peptide, calcitonin, amylin) increase the rate of active Na+, K+ transport by 60-120% within a few minutes. This leads to a decrease in intracellular Na+ and hyperpolarization. In isolated muscles, where contractility is inhibited by high extracellular K(+)- such agents produce rapid force recovery. which is entirely suppressed by ouabain and closely correlated to the stimulation of K+ uptake and the decline in intracellular Na+. The observations support the conclusion that the Na+, K+ pump plays a central role in the acute recovery and maintenance of excitability during contractile activity.
Animals↗
Characterization of bumetanide-sensitive Na+ and K+ transport in rat skeletal muscle.
In isolated rat soleus muscle an average of 23% of the total 22Na influx was found to be suppressible by bumetanide (K0.5 = 0.1 mM) and furosemide (K0.5 = 1 mM), whereas the influx and efflux of 42K were not affected. In extensor digitorum longus muscle, around 25% of the total 22Na influx was suppressible by bumetanide (1 mM). In the presence of ouabain, both diuretics decreased net intracellular accumulation of Na+, but caused no change in K+ content. In extensor digitorum longus (but not in soleus), bumetanide-suppressible 22Na influx was stimulated by increasing extracellular osmolarity with the bumetanide having no effect on 42K influx. Bumetanide-suppressible Na+ influx was almost abolished in Cl(-)-free buffer, but was unaffected by the omission of K+. In rat soleus, the inhibitory effects of bumetanide, amiloride and tetrodotoxin on 22Na influx were found to be additive. The results indicate that a NaCl cotransport system is present in both fast- and slow-twitch skeletal muscles, and may participate in volume regulation. Due to the large pool of muscle cells, activation of NaKCl2 cotransport is likely to entail the hazards of hypokalemia. The advantage of exerting volume control via NaCl cotransport is that this risk can be avoided.
Animals↗
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(-5) M 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 x 10(-6) M). 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.
Animals↗
Role of thyroid hormones in early postnatal development of skeletal muscle and its implications for undernutrition.
Energy intake profoundly influences many endocrine axes which in turn play a central role in development. The specific influence of a short period of mild hypothyroidism, similar to that induced by undernutrition, in regulating muscle development has been assessed in a large mammal during early postnatal life. Hypothyroidism was induced by providing methimazole and iopanoic acid in the feed of piglets between 4 and 14 d of age, and controls were pair-fed to the energy intake of their hypothyroid littermates. Thyroid status was evaluated, and myofibre differentiation and cation pump concentrations were then assessed in the following functionally distinct muscles: longissimus dorsi (l. dorsi), soleus and rhomboideus. Reductions in plasma concentrations of thyroxine (T4; 32%, P < 0.01), triiodothyronine (T3; 48%, P < 0.001), free T3 (58%, P < 0.001) and hepatic 5'-monodeiodinase (EC 1.11.1.8) activity (74%, P < 0.001) occurred with treatment. Small, although significant, increases in the proportion of type I slow-twitch oxidative fibres occurred with mild hypothyroidism, in l. dorsi (2%, P < 0.01) and soleus (7%, P < 0.01). Nuclear T3-receptor concentration in l. dorsi of hypothyroid animals compared with controls increased by 46% (P < 0.001), a response that may represent a homeostatic mechanism making muscle more sensitive to low levels of circulating thyroid hormones. Nevertheless, Na+, K(+)-ATPase (EC 3.6.1.37) concentration was reduced by 15-16% in all muscles (l. dorsi P < 0.05, soleus P < 0.001, rhomboideus P < 0.05), and Ca(2+)-ATPase (EC 3.6.1.38) concentration was significantly reduced in the two slow-twitch muscles: by 22% in rhomboideus (P < 0.001) and 23% in soleus (P < 0.05). It is concluded that during early postnatal development of large mammals a period of mild hypothyroidism, comparable with that found during undernutrition, induces changes in myofibre differentiation and a down-regulation of cation pumps in skeletal muscle. Such changes would result in slowness of movement and muscle weakness, and also reduce ATP hydrolysis with a concomitant improvement in energetic efficiency.
Animals↗
Involvement of Tyr24 and Trp108 in substrate binding and substrate specificity of glycolate oxidase.
Tyr24 and Trp108 are located in the active site of spinach glycolate oxidase. To elucidate their function in substrate binding and catalysis, they were replaced by phenylalanine and serine, respectively. The [Y24F]glycolate oxidase mutant enzyme showed a tenfold higher Km value for glycolate. L-lactate and DL-2-hydroxybutyrate also showed higher Km values, however, the substrate specificity was unchanged as compared to the wild-type enzyme (Km increases in the order glycolate < DL-2-hydroxybutyrate < L-lactate < L-mandelate). The turnover number and the rate of reduction, found to be rate limiting in catalysis, were only slightly affected by the deletion of the hydroxyl group. These findings suggest that Tyr24 is mostly involved in substrate binding. The spectral features of the [Y24F]glycolate oxidase suggest that a fraction (50-80%) of the protein bears a flavin N(5) adduct instead of the oxidized cofactor. Crystals obtained from the isolated [Y24F]glycolate oxidase mutant protein allowed the determination of the three-dimensional structure. Although the structure was low resolution (0.3 nm), it is evident that the structure determined is that of the N(5) adduct species. In addition to the lacking hydroxyl group of Tyr24, we also observed movements of the amino acid side chains of Arg164 and Trp108, the latter replacing a water molecule in the substrate-binding pocket. Other features predominantly found in the class of flavoprotein oxidases, such as stabilization of the covalent N(5)-sulfite adduct and of the paraquinoid form of 8-mercapto-FMN, were found to be conserved. [W108S]Glycolate oxidase, in contrast, showed dramatic effects on both the Km of substrates as well as the turnover number. The Km for glycolate was increased some hundred fold and the turnover number was decreased 500-fold. In addition, it was found that the higher homologs of glycolate, L-lactate and DL-2-hydroxybutyrate had turnover numbers similar to those found with the wild-type enzyme, although the Km values also increased dramatically. These results indicate that Trp108 is of major importance in catalysis and that this residue is involved in determining the substrate specificity of glycolate oxidase.
Alcohol Oxidoreductases↗
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 4mM to 8-14mM 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 8-9mM, a further small increase in [K+]0 to 11-14mM resulted in a large reduction of force. Tetanus depression at 11mM [K+]0 was increased when using weaker stimulation pulses and decreased with stronger pulses. Whereas the tetanic force/resting membrane potential (EM) relation showed only moderate force depression with depolarization from -74 to -62mV, a large reduction of force occurred when EM fell to-53mV. 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 11mM [K+]0. Salbutamol, insulin, or calcitonin gene-related peptide all stimulated the Na+-K+ pump in muscles exposed to 11mM [K+]0 and induced an average 26-33% recovery of tetanic force. When using stimulation pulses of 0.1ms, 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 of EM (1.3mV). 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.
Animals↗
Measurement of the total concentration of functional Na+, K(+)-pumps in rumen epithelium.
Using the technique of vanadate-facilitated [3H]ouabain binding we have developed a simple and reliable assay for measuring the concentration of [3H]ouabain binding sites in small fresh or frozen biopsies of rumen epithelium papillae. In bovine and ovine rumen epithelium obtained from the cranio-ventral rumen sac the concentration of [3H]ouabain binding sites was 1.6-4.9 nmol g dry wt-1 (n = 32) and 3.7-5.2 nmol g dry wt-1 (n = 6), respectively. When incubated in oxygenated Krebs-Ringer bicarbonate buffer fresh biopsies of rumen epithelium maintained a high K+ and low Na+ content for at least 6 h. Na+ loading of the biopsies induced about 20-fold increase of the Na+, K(+)-pump activity based on measurement of ouabain suppressible net [86Rb+] influx. The ouabain suppressible net influx of [86Rb+] measured in Na+ loaded biopsies showed a close correlation to the [3H]ouabain binding capacity (r = 0.80, P < 0.01) and corresponded to 47 +/- 2% (n = 9) of the theoretical maximum flux rate. The ouabain suppressible net influx of K+ and [86Rb+] were linearly related (r = 0.73; P < 0.001). The net Na+ efflux was 1.21 times the net K+ influx. It is concluded that rumen epithelium has a large capacity for active Na+/K+ transport and that there is agreement between the concentration of [3H]ouabain binding sites in the epithelium and the ouabain suppressible rate of net [86Rb+] influx in Na+ loaded biopsies in spite of some uncertainty about the maximum turnover number of the Na+, K(+)-pump in rumen epithelium.
Absorption↗
Insulin-like growth factor I stimulates active Na(+)-K+ transport in rat soleus muscle.
The functional homology between insulin and insulin-like growth factor I (IGF-I) comprises effects on growth and glucose metabolism. Because insulin stimulates the Na(+)-K+ pump, IGF-I might exert a similar effect. We show here that IGF-I increases 42K and 86Rb uptake and the efflux of 22Na in isolated rat soleus muscle. This leads to a significant decrease (21-55%, P < 0.001) in intracellular Na+ and a small increase in intracellular K+. In extensor digitorum longus (EDL) muscle, similar effects were observed. The stimulation of K+ uptake and the reduction in intracellular Na+ in the soleus were blocked by ouabain, indicating that they reflect an acute stimulation of active Na(+)-K+ transport. This conclusion was further supported by the observation that the [3H]ouabain binding rate was significantly increased by IGF-I. IGF-I increased ouabain-suppressible 42K or 86Rb uptake by 56 and 54%, respectively. The effects of IGF-I and epinephrine on ouabain-suppressible 86Rb influx in rat soleus were additive, whereas the effects of insulin and IGF-I were similar and nonadditive. The effects of IGF-I were seen down to a concentration of 10(-8) M, which is unlikely to stimulate the insulin receptor, and it is therefore plausible that IGF-I exerts its effect on Na(+)-K+ transport through its own receptor. IGF-I may play a role in the maintenance of muscle Na+ and K+ contents also in vivo, especially in patients treated with IGF-I.
Animals↗
Televised violence: a Japanese, Spanish, and American comparison.
In Japan, Spain, and the USA during one week acts of violence (verbal and nonverbal) were rated on the Index of Television Violence by 3 raters in each country. In all three countries televised violence was more likely depicted in interpersonal conflict than against property. Fewer scenes of physical or fatal injury appeared in Japan than in the other two countries. Other observations were made. Replication is urged.
Adult↗
86Rb is not a reliable tracer for potassium in skeletal muscle.
For technical reasons, 86Rb is frequently preferred to 42K as a tracer for K+. Systematic comparisons of the two isotopes, however, are rarely done. In this paper we compare the transport of 42K and 86Rb in rat and mouse soleus muscle and in rat erythrocytes. Ouabain-suppressible K+ uptake in rat soleus was the same whether measured with 42K or 86Rb, both when stimulated by insulin, salbutamol and calcitonin-gene-related peptide (CGRP), and when inhibited by graded concentrations of ouabain. Control experiments with rat erythrocytes, where Na(+)-K(+)-Cl- co-transport has earlier been demonstrated, showed closely similar inhibitory effects of bumetanide on 42K and 86Rb uptake. In contrast, bumetanide produced no significant change in 42K uptake of rat and mouse soleus muscle, but clearly inhibited 86Rb uptake at concentrations down to 10(-7) M (P < 0.001). Whereas the addition of 150 mM NaCl had no effect on 42K uptake in rat soleus, 86Rb uptake, and in particular the bumetanide-suppressible component, was markedly increased by this addition. The inhibitory effect of bumetanide on 86Rb uptake gives rise to the false impression that skeletal muscle contains a NaKCl2 co-transport system. Efflux studies showed that the fractional loss of 42K from rat soleus muscle is 2.3 times larger than that of 86Rb. Salbutamol and CGRP increased 86Rb efflux, but inhibited 42K efflux. This implies that for studies of K+ efflux and bumetanide-sensitive K+ transport, 86Rb is not even an acceptable tracer for the detection of qualitative changes. Control experiments with 42K are essential in any characterization of unknown K+ transport processes.
Albuterol↗
Effects of intensified endurance training on the concentration of Na,K-ATPase and Ca-ATPase in human skeletal muscle.
Thirty-nine moderately endurance trained males increased their normal training programme of 2.2 h week-1 with an average training intensity of 65% of maximum heart rate (HRmax) to 2.7 h week-1 and a mean intensity of 78% of HRmax. Performance tests and measurements of the total concentrations of Na,K-ATPase (3H-ouabain binding) and Ca-ATPase, fibre type distribution and fibre area were performed in biopsies from the vastus lateralis muscle before and after increased training. The 6 weeks of training elevated VO2max from 54.9 +/- 3.1 to 58.3 +/- 3.0 ml O2 min-1 kg-1 (P < 0.0001). Exercise time to exhaustion at 86% of VO2max (pre-training) increased from 35 +/- 8 to 61 +/- 17 min (P < 0.0001). The concentration of Ca-ATPase was unaffected by the intensified training (6.74 +/- 1.03 vs. 6.68 +/- 1.07 nmol g wet wt-1), but the concentration of Na,K-ATPase increased from 307 +/- 43 to 354 +/- 59 pmol g wet wt-1 (P < 0.0001). The relative distribution of FT-fibres was correlated with the concentration of Ca-ATPase (r = 0.72, P < 0.0001). The data support the view that intensive training induces an upregulation of the concentration of skeletal muscle Na,K-ATPase, but no change in the total capacity for reaccumulation of Ca2+ into the SR. There was no correlation between the concentrations of Na,K-ATPase, Ca-ATPase and indices of endurance performance.
Adult↗
The Na+,K(+)-pump and muscle contractility.
In skeletal muscle, the excitation induced influx of Na+ and efflux of K+ may be sufficient to exceed the activity or even the capacity of the available Na+,K(+)-pumps. This leads to a rise in intracellular Na+ and extracellular K+. Both events interfere with excitability and may present important limitations for the continuation of contractile activity. Furthermore, inhibition of the Na+,K(+)-pump or reduction of the concentration of functional Na+,K(+)-pumps decrease excitability and the maintenance of force during continued stimulation. Conversely, in muscles where contractile force is inhibited by exposure to high extracellular K+, acute stimulation of the Na+,K(+)-pump with catecholamines, CGRP or insulin leads to a rapid recovery of force. The large passive fluxes of Na+ and K+ associated with excitation constitute the major drive on the activity of the Na+,K(+)-pump, giving rise to up to 20-fold stimulation of the transport rate. In keeping with this, training induces an upregulation of the total concentration of Na+,K(+)-pumps in skeletal muscle. The activity and the capacity of the Na+,K(+)-pump are important limiting factors determining the maintenance of excitability and contractile performance.
Animals↗
Excitation-induced activation of the Na(+)-K+ pump in rat skeletal muscle.
The stimulating effect of excitation on the Na(+)-K+ pump was characterized in measurements of 22Na efflux, intracellular Na+ content, 86Rb influx, and [3H]ouabain binding in isolated rat soleus muscle. Direct stimulation (10 V, 1 ms, 2 Hz) rapidly increased 22Na efflux and 86Rb influx about twofold. These effects were blocked by tetracaine and ouabain, were not associated with any significant increase in intracellular Na+, and could not be attributed to a rise in extracellular K+. The stimulation of 22Na efflux was unaffected by tubocurarine, dantrolene, trifluoperazine, or bumetanide. Stimulation at 2 Hz increased the rate of [3H]ouabain binding by approximately 120% within 1 min, indicating an early specific activation of the Na(+)-K+ pump. Stimulation at 60 Hz for 10 s increased intracellular Na+ content by 58%. Reextrusion of Na+ was complete in 2 min and could be prevented by ouabain (10(-4) M) or by cooling to 0 degrees C. It is concluded that, in rat soleus muscle, excitation leads to a rapid and pronounced (up to 15-fold) stimulation of the Na(+)-K+ pump, even at modest increases in intracellular Na+. This activation mechanism may be essential for the maintenance of transmembrane Na(+)-K+ gradients and prompt recovery of excitability during contractile activity.
Animals↗
Roles of skeletal muscle morphology and activity in determining Na(+)-K(+)-ATPase concentration in young pigs.
It has been demonstrated previously that the Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) concentration (total concentration of [3H]ouabain-binding sites = maximal [3H]ouabain-binding capacity, Bmax) in longissimus dorsi muscle is higher in young pigs living in a cold than in a warm environment, with the effect being even greater in those on a low rather than a high energy intake in the cold. The importance of thyroid status, myofiber type and size, and muscular activity in determining the concentration of Na(+)-K(+)-ATPase has therefore been assessed in longissimus dorsi, semitendinosus, and soleus muscles from 8-wk-old littermate pigs, which had been living 35 or 10 degrees C on either a high or a low energy intake for 3-4 wk. Changes in neither plasma thyroid hormone concentrations nor myofiber type could account for the greater Bmax at 10 vs. 35 degrees C observed in all three muscles; instead, it was possibly related to the greater muscular activity associated with shivering in the cold. More than 50% of the increase in Bmax on the low compared with the high intake in the cold could be attributed to the smaller fiber size and hence greater membrane area in animals on the low intake, and it is concluded that membrane surface area must be assessed in studies of membrane-bound enzymes.
Animals↗
[Acetylsalicylic acid and pre-eclampsia/growth retardation].
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Aspirin↗