The effects of ATP, inorganic phosphate, protons, and lactate on isolated myofibrillar ATPase activity.

The purpose of this study was to examine the effects of lactate, protons, inorganic phosphate, and ATP on myofibrillar ATPase activity. Myofibrils were isolated from carp (Cyprinius carpio L.) fast-twitch white muscle, and myofibrillar ATPase activities were assessed under maximal activating calcium levels (pCa 4.0) at 10 degrees C in reaction media containing metabolic profiles similar to those seen in fatiguing muscles. The Ca(2+)-activated ATPase activity was assessed by an ATP regenerating assay that coupled the myofibrillar ATPase to pyruvate kinase and lactate dehydrogenase. This assay allowed the effects of ATP, inorganic phosphate, protons, and lactate on myofibrillar ATPase activity to be assessed. The coupled assay was found to give similar myofibrillar ATPase kinetics, with the exception of higher maximal activities, to those seen with a standard end-point assay. Myofibrillar ATPase activity was depressed by 35% when ATP concentrations were lowered to 2.5 mM. Lowering ATP levels to 0.5 mM reduced the myofibrillar ATPase activities by 85%. Lactate had no effect on myofibrillar ATPase activities. Inorganic phosphate levels up to about 20 mM significantly decreased the myofibrillar ATPase activities, after which further increases in inorganic phosphate content had minimal effects. The changes in ATPase activities were related to total inorganic phosphate, not to the content of diprotonated inorganic phosphate. Myofibrillar ATPase activity was highest at pH 7.5 and lowest at pH 6.0. The interactive effects of low ATP, decreased pH, and high inorganic phosphate levels were not additive, giving similar decreases in activity to those produced by increased inorganic phosphate levels alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of fasting and refeeding on serum parathormone and calcitonin concentrations in young and old male rats.

Although fasting and refeeding reveal the existence of age-related changes in carbohydrate and lipid metabolism, the effects of aging on mineral metabolism in refed animals are unknown. We therefore investigated hormonal regulation of calcium metabolism in young (4 months) and old (26 months) male rats fasted for 48 hours and then refed for 4 or 24 hours. Serum concentrations of total and ionized calcium and parathormone were similar in control young and old rats. Serum calcitonin level was higher, and the concentrations of albumin and inorganic phosphate and alkaline phosphatase activity were lower in fed old rats. In young fasted rats, the serum ionized and total calcium was decreased, and phosphate concentration was increased. In old rats, fasting resulted in the increase of serum parathormone level. Fasting reduced serum alkaline phosphatase activity to a similar extent in both age groups. In young rats, refeeding for 24h normalized serum calcium and phosphate levels and alkaline phosphatase activity, and decreased serum concentrations of PTH and calcitonin. In old refed rats, serum calcitonin concentration was raised by 77% compared to fed or fasted animals, whereas parathormone levels were normalized. Our results indicate that old fasted or refed rats maintain normal serum calcium concentration in a different way than young animals, possibly through the increase in serum levels of parathormone and/or calcitonin. Thus, dietary manipulations such as fasting and refeeding constitute an interesting model for the investigation of the effects of aging on the hormonal regulation of serum calcium level.     

Influence of potassium and calcium ions on the phosphatidylinositol and phosphatidylcholine metabolism in rat fibroblasts after growth stimulation by calf serum.

In secondary cultures of embryonic rat fibroblasts which were arrested in G1 (G0) by serum depletion and subsequently triggered into the cell cycle by readdition of growth factors isolated from fetal calf serum the influence of the potassium and calcium concentrations in the medium on phosphatidylinositol and phosphatidylcholine metabolism was investigated. The incorporation of inorganic [32P]phosphate into phosphatidylinositol is dependent on the potassium content of the culture medium. The specific activity of 32P in phosphatidylinositol is increased at K+ concentrations between 0.1 and 1 mM. Also calcium (between 0.01 and 2 mM) slightly stimulates phosphatidylinositol metabolism. Also the incorporation of myo-[3H]inositol is increased at potassium concentrations between 0.2 and 1 mM, whereas calcium is slightly inhibitory. The labelling of phosphatidylcholine with either [32P]phosphate or [3H]choline is not dependent on the potassium and calcium concentrations of the culture medium. Moreover, the phospholipid metabolism of permanently growing epithelioid and fibroblastoid cells lines, which were investigated, is considerably less dependent on the K+ and Ca2+ ions.     

Analysis of phosphate metabolites, the intracellular pH, and the state of adenosine triphosphate in intact muscle by phosphorus nuclear magnetic resonance.

31P nuclear magnetic resonance spectra recorded from intact muophosphate, and the sugar phosphates. Quantitation of these metabolites by 31P nuclear magnetic resonance was in good agreement with values obtained by chemical analyses. The spectra obtained from various muscles showed considerable variation in their phosphorus profile. Thus, differences could be detected between (a) normal and diseased muscle; (b) vertebrates and invertebrates; (c) different species of the same animal. The time course of change in phosphate metabolites in frog muscle showed that ATP level remains unchanged until phosphocreatine is nearly depleted. Comparative studies revealed that under anaerobic conditions the Northern frog maintains its ATP content for 7 hours, while other types of amphibian, bird, and mammalian muscles begin to show an appreciable decay in ATP after 2 hours. Several lines of evidence indicated that ATP forms a complex with magnesium in the muscle water: (a) the phosphate resonances of ATP in the muscle were shifted downfield as compared to those in the alkaline earth metal-free perchloric acid extract of the muscle; (b) the coupling constants of ATP measured in various live muscles closely corresponded to those for MgATP in a solution resembling the composition of the muscle water; (c) in the muscle the gamma-phosphate group of ATP exhibited no shift change over a period of 10 hours under conditions where resonances of other phosphate compounds could be titrated. This behavior is similar to that of MgATP in model solutions in the physiological pH range, and it is different from that of CaATP. The chemical shifts of the phosphate metabolites were determined in several relevant solutions as a function of pH. Under all conditions only inorganic orthophosphate showed an invariant titration curve. From the chemical shift of inorganic phosphate observed during aging of intact muscle the intracellular pH of frog muscle was estimated to be 7.2.     

The effect of heat-stable extract from bovine sublingual salivary gland on serum cholesterol and triglyceride levels in normal and hypercholesterolemic rabbits

An extract of bovine sublingual glands (SLF3) reduced the serum cholesterol levels of normal rabbits 34.6% and serum triglyceride levels 19.6% when injected intraperitoneally (i.p.) at 20 mg/kg body wt on alternate days for 7 days. SLF3 also reduced serum cholesterol levels 69.0% and triglyceride levels 46.5% in hypercholesterolemic rabbits, while in similar rabbits injected with a control muscle tissue extract, the rate of decrease in serum cholesterol levels was 33.3% and triglyceride levels 26.7%.     

Phospholipid metabolism of monkey smooth muscle cells grown in hyperlipemic serum.

The phospholipid content and synthesis of monkey smooth muscle cells grown in tissue culture with normal or hyperlipemic monkey serum were examined. The pattern of incorporation of radioactively labeled inorganic phosphate into the phospholipids of these cells was measured using a 4 h pulse of 32P. Phosphatidylcholine and phosphatidylinositol were the predominant phospholipids labeled. Although phosphatidylcholine constituted 45% of the cellular phospholipid, phosphatidylinositol had the highest specific activity. Exposure of the smooth muscle cells to hyperlipemic monkey serum did not alter the phospholipid content, composition or synthesis of these cells. The total phospholipid content of the smooth muscle cells was independent of the concentration of lipid in the media. The distribution of 32P into the phospholipids of monkey alveolar macrophages, L-cell mouse fibroblasts, and segments of the intima-media from monkey aortas is reported.     

Regulation of glycolytic flux in an energetically controlled cell-free system: the effects of adenine nucleotide ratios, inorganic phosphate, pH, and citrate

A soluble extract from rat skeletal muscles has been used with purified mitochondrial ATPase (F₁) to develop steady states with respect to glycolytic flux, the concentrations of glycolytic intermediates and inorganic phosphate, and the concentrations and ratios of adenine nucleotides. Incubations were carried out in media resembling the ionic composition in the cell cytoplasm, in an attempt to evaluate the quantitative contributions of various effectors to the overall control mechanism under simulated in vivo conditions. The primary control reaction of glycolytic flux under the conditions studied could be identified with phosphofructokinase, followed by secondary control of the reaction catalyzed by hexokinase. Glycolytic flux was increased with increasing pH over the range 6.6–7.6, both in the absence and presence of ATPase. Without other added effectors, the glycolyzing extract maintained an ATP/ADP ratio of about 50 in the pH range 7.0–7.6, and phosphofructokinase was incompletely suppressed. Addition of increasing amounts of ATPase markedly stimulated glycolytic flux coincident with lowered steady-state ATP/ADP ratios, and decreased accumulation of hexose monophosphates. Control of flux by the ATP/ADP ratio (and simultaneously altered AMP concentration) was less effective if pH (7.3 to 7.6) or phosphate concentration (2 to 20 mm) was increased. Flux through phosphofructokinase was controlled principally when the ATP/ADP ratios were varied in the range between > 50 and 15. The inhibitory effect of citrate was evaluated. Suppression of glycolytic flux and accumulation of hexose monophosphates were dependent on incubation conditions. If the pH was 7.3 or less, and the phosphate concentration low (2 mm), flux through phosphofructokinase was significantly suppressed even at citrate concentrations less than 50 μm. Simultaneous decrease in the steady-state ATP/ADP ratio and elevation of AMP was ineffective in reversing this inhibition. At higher pH and, more dramatically, when the phosphate concentration was increased, sensitivity to citrate inhibition was markedly diminished. These data, taken together with studies of respiratory control with isolated mitochondria (21., 24.), J. Biol. Chem.250, 2275–2282) strongly suggest that adenine nucleotide control of both glycolysis and respiration is exerted when the ratio of free nucleotides (not protein bound) in the cytosol is in the range of 15 to > 50. The data further suggest that citrate plays an important role in the regulation of glycolysis in muscle when the ATP/ADP ratio is high (and the phosphate concentration is correspondingly low), but that this inhibition is overcome by liberation of inorganic phosphate during muscle contraction.     

Thyroid hormones and muscle metabolism in dogs

Muscle contents of ATP, ADP, AMP, creatine phosphate and creatine as well as glycogen, some glycolytic intermediates, pyruvate and lactate were compared in the intact, thyroidectomized and triiodothyronine (T3) treated dogs under resting conditions. After thyroidectomy muscle glycogen, glucose 1-phosphate and glucose 6-phosphate contents were significantly elevated while in T3-treated animals these variables were decreased in comparison with control dogs. Muscle free glucose was not altered by thyroidectomy but T3 treatment significantly increased its content. Muscle lactate content was elevated both in hypo- and hyperthyroid animals. Muscle ATP and total adenine nucleotide contents were significantly increased in hyperthyroid dogs while no differences were found between the three groups in the muscle creatine phosphate content. It is assumed that in T3-treated animals carbohydrate catabolism is enhanced in the resting skeletal muscle in spite of high tissue ATP content. Muscle metabolite alterations in hypothyroid dogs seem to reflect the hypometabolism accompanied by a diminished rate of glycogenolysis with inhibited rate of pyruvate oxidation or decreased rate of lactate removal from the cells.     

Influence of Inorganic Phosphate on Photosynthesis of Wheat Chloroplasts: II. RIBULOSE BISPHOSPHATE CARBOXYLASE ACTIVITY

Isolated wheat chloroplasts were pre-incubated in the dark inthe presence of various concentrations of inorganic phosphatewith or without carbon dioxide, oxaloacetate, glycerate, and3-phosphoglycerate. The effect of subsequent illumination onphotosynthetic oxygen evolution, ribulose bisphosphate carboxylaseactivity, ATP content, and ribulose bisphosphate content wasinvestigated. Inorganic phosphate had little effect on ribulosebisphosphate carboxylase activity in darkness or during theinitial phase of illumination, but it prevented the declinein activity that occurred during later stages of illumination,when photoreduction of CO₂ was decreasing in rate. Additionof inorganic phosphate to chloroplasts illuminated without phosphaterestored the ribulose bisphosphate carboxylase activity, increasedthe ATP, and decreased the ribulose bisphosphate in the organelles.The responses to CO₂, oxaloacetate, glycerate, and 3-phosphoglyceratesuggest that the decreased activity of ribulose bisphosphatecarboxylase during photosynthesis results from ATP consumption. Purified ribulose bisphosphate carboxylase was activated byinorganic phosphate, but this activation did not occur in thepresence of ATP. ATP inhibited ribulose bisphosphate carboxylasewhen it was present in combination with various photosyntheticmetabolites. Inactivation of ribulose bisphosphate carboxylase in chloroplasts,illuminated in the absence of inorganic phosphate, is not dueto lack of activation by inorganic phosphate or ATP. It mayresult from decreased stromal pH. Key words: Ribulose bisphosphate carboxylase, Chloroplasts, Wheat, Phosphate, ATP     

Regulation of photosynthetic carbon metabolism during phosphate limitation of photosynthesis in isolated spinach chloroplasts

Intact chloroplasts isolated from spinach were illuminated in the absence of inorganic phosphate (Pi) or with optimum concentrations of Pi added to the reaction medium. In the absence of Pi photosynthesis declined after the first 1–2 min and was less than 10% of the maximum rate after 5 min. Export from the chloroplast was inhibited, with up to 60% of the ¹⁴C fixed being retained in the chloroplast, compared to less than 20% in the presence of Pi. Despite the decreased export, chloroplasts depleted of Pi had lower levels of triose phosphate while the percentage of total phosphate in 3-phosphoglycerate was increased. Chloroplast ATP declined during Pi depletion and reached dark levels after 3–4 min in the light without added Pi. At this point, stromal Pi concentration was 0.2 mM, which would be limiting to ATP synthesis. Addition of Pi resulted in a rapid burst of oxygen evolution which was not initially accompanied by net CO₂ fixation. There was a large decrease in 3-phosphoglycerate and hexose plus pentose monophosphates in the chloroplast stroma and a lesser decrease in fructose-1,6-bisphosphate. Stromal levels of triose phosphate, ribulose-1,5-bisphosphate and ATP increased after resupply of Pi. There was an increased export of ¹⁴-labelled compounds into the medium, mostly as triose phosphate. Light activation of both fructose-1,6-bisphosphatase and ribulose-1,5-bisphosphate carboxylase was decreased in the absence of Pi but increased following Pi addition.It is concluded that limitation of Pi supply to isolated chloroplasts reduced stromal Pi to the point where it limits ATP synthesis. The resulting decrease in ATP inhibits reduction of 3-phosphoglycerate to triose phosphate via mass action effects on 3-phosphoglycerate kinase. The lack of Pi in the medium also inhibits export of triose phosphate from the chloroplast via the phosphate transporter. Other sites of inhibition of photosynthesis during Pi limitation may be located in the regeneratige phase of the reductive pentose phosphate pathway.Abbreviations FBP Fructose-1,6-bisphosphate - FBPase Fructose-1,6-bisphosphatase - MP Hexose plus pentose monophosphates - PGA 3-phosphoglycerate - Pi inorganic orthophosphate - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase - TP Triose Phosphate     

The Co-effect of Cordyceps sinensis and Strontium on Osteoporosis in Ovariectomized Osteopenic Rats

The co-effect of Cordyceps sinensi (CS; caterpillar fungus) and strontium on ovariectomized osteopenic rats was studied in this paper. After the rats were treated orally with CS, strontium (SR), and CS rich in strontium (CSS), respectively, the urine calcium, plasma calcium, plasma phosphorus, bone mineral content, mechanical testing, and the mass of uterus, thymus, and body were examined. Both CSS and SR have a positive effect on mechanical strength and mineral content of ovariectomized osteopenic rats. However, femoral neck strength in the CSS-treated group was higher than those in the SR-treated groups. CSS and SR significantly decreased urinary calcium excretion and plasma total calcium and inorganic phosphate concentrations. On the contrary, CS and CSS significantly increased weights of atrophic uteri and weights of body and also decreased the thymus mass in animals, whereas SR did not exhibit any such effects. Our experiments have demonstrated that CSS possess a preferable effect against the decrease of bone strength and bone mineral mass caused by osteoporosis. It was caused by the co-effect of CS and strontium. The mechanism of it includes decreases bone resorption, increases bone formation, increases in body weight, and enhances 17β-estradiol-producing as well as enhancing the immune functions in animals. The data provide an important proof of concept that CSS might be a new potential therapy for the management of postmenopausal osteoporosis in humans.     

Lipopolysaccharide markedly changes glucose metabolism and mitochondrial function in the longissimus muscle of pigs

Most previous studies on the effects of lipopolysaccharide (LPS) in pigs focused on the body’s immune response, and few reports paid attention to body metabolism changes. To better understand the glucose metabolism changes in skeletal muscle following LPS challenge and to clarify the possible mechanism, 12 growing pigs were employed. Animals were treated with either 2 ml of saline or 15 µg/kg BW LPS, and samples were collected 6 h later. The glycolysis status and mitochondrial function in the longissimus dorsi (LD) muscle of pigs were analyzed. The results showed that serum lactate content and NADH content in LD muscle significantly increased compared with the control group. Most glycolysis-related genes expression, as well as hexokinase, pyruvate kinase and lactic dehydrogenase activity, in LD muscle was significantly higher compared with the control group. Mitochondrial complexes I and IV significantly increased, while mitochondrial ATP concentration markedly decreased. Significantly increased calcium content in the mitochondria was observed, and endoplasm reticulum (ER) stress has been demonstrated in the present study. The results showed that LPS treatment markedly changes glucose metabolism and mitochondrial function in the LD muscle of pigs, and increased calcium content induced by ER stress was possibly involved. The results provide new clues for clarifying metabolic diseases in muscle induced by LPS.     

31P-NMR studies of phosphate metabolites in intact red and white swimming muscles of cod (Gadus morhua L.)

31P-NMR was used to characterise intracellular phosphate pools and their post mortem changes at 7 degrees C in intact red and white cod muscles under anaerobic conditions. A total phosphate content of 55 and 60 mM was observed in red and white muscle, respectively. The concentration of P-creatine was 14 mM in the white and 9 mM in the red muscle, while that of inorganic phosphate, Pi (30 mM), ATP (9 mM), and sugar phosphate (5 mM) were similar in both muscles. During the first 90 min after death, the decrease in P-creatine showed a first order breakdown with a concomitant stoichiometric increase in Pi content, whereas the ATP and sugar phosphate remained the same. The intracellular pH decreased from 7.4 to 7.3 in this period. The steady-state rate constant of myosin ATPase was 0.0054 and 0.0022/min for red and white muscles, respectively. Individuals kept under diminished oxygen tension prior to being killed, showed a reduced P-creatine level in both muscles.     

The influence of inorganic phosphate and ATP on the kinetics of bovine heart muscle pyruvate kinase

Summary The mechanism of activation by inorganic phosphate and ATP of cardiac muscle pyruvate kinase was studied with the aid of steady-state kinetics. The enzyme was purified to homogeneity to a final specific activity of 400 units/ mg (phosphate buffer, pH 7.6, 25 °C). At pH 7.6 the enzyme displays Michaelis-Menten kinetics with respect to both its substrates, phosphoenolpyruvate and ADP. Substrate kinetic constants are: app.K_m(phosphoenolpyruvate) –0.04 mM, app.K_m(ADP) =0.22 mM. Under the conditions used in the standard assay the specific activity is greatly enhanced by inorganic phosphate (50 mM) or ATP (2.5 mM). Each of these modifiers, acting separately, increases the V_max without seriously affecting Michaelis constants and Hill coefficients. In the presence of both Pi and ATP, only a decrease in V_max was observed.The kinetics of activation by inorganic phosphate of pyruvate kinase was examined. Studying the effect of varying concentrations of Pi on the initial rate we obtained a hyperbolic saturation curve with the app. K_m(P_i) = 20 mM and V_max = 167 units/ mg. The evidence is presented that inorganic phosphate is a substrate for a side reaction catalyzed by cardiac pyruvate kinase. It is shown that in the presence of pyruvate, inorganic phosphate and ATP in the assay system, P_i is incorporated into acid-labile products of this reaction, inorganic pyrophosphate being one of them.These findings indicate the existence of an alternative reaction catalyzed by pyruvate kinase by which energy may be stored in the form of inorganic pyrophosphate.Abbreviations PEP phosphoenolpyruvate - Pi inorganic phosphate - TEA triethanolamine - EDTA ethylenediaminetetraacetate     

Triosephosphates modulate leaf mitochondrial phosphorylation by inhibition and uncoupling of electron transport

The effect of TP (triosephosphates:glyceraldehyde-3 phosphate, GAP, +dihydroxyacetone phosphate, DHAP) on respiration, phosphorylation and matrix ATP/ADP ratios of isolated oat mesophyll mitochondria was investigated. With both malate and NADH, a 50% inhibition of state 3-phosphorylation was induced by about 15 to 20 millimolar GAP and 30 to 40 millimolar DHAP. However, the nature of the inhibition appeared to be different with the two respiratory substrates. In the presence of NADH, TP did not inhibit the rate of state 3 (addition of ADP) O₂ consumption. In fact, depending on concentration, TP gradually increased the rates measured without ADP towards those seen under state 3, acting as uncouplers. When malate was the substrate for respiration, state 3 rates were decreased. The effect was comparable to that of rotenone and could be abolished by the addition of NADH. These observations indicate a dual action of TP: inhibition of electron transport around site I and uncoupling. In any case, the intramitochondrial ATP/ADP ratio decreased upon addition of TP. The effective TP concentrations as well as the changes in mitochondrial ATP/ADP ratios were comparable to results on changes of compartmental pool sizes of adenylates and other metabolites during dark/light transition of oat mesophyll protoplasts (R. Hampp, M. Goller, H. Füllgraf, and I. Eberle 1985 Plant Cell Physiol 24: 99). The possible role of TP in the regulation of mitochondrial respiration in the light, as well as modes of interference, are discussed.     

The influence of phosphate deficiency on photosynthesis, respiration and adenine nucleotide pool in bean leaves

The decrease in inorganic phosphate (Pi) content of 10-d-old Phaseolus vulgaris L. plants did not affect rates of photosynthesis (PN) and respiration (RD), leaf growth, and adenylate concentration. Two weeks of phosphate starvation influenced the ATP content and leaf growth more than PN and RD. The ATP concentration in the leaves of 15- and 18-d-old phosphate deficient (-P) plants after a light or dark period was at least half of that in phosphate sufficient (+P, control) plants. Similar differences were found in fresh and dry matter of leaves. However, PN declined to 50 % of control in 18-d-old plants only. Though the RD of -P plants (determined as both CO2 evolution and O2 uptake) did not change, an increased resistance of respiration to KCN and higher inhibition by SHAM (salicylhydroxamic acid) suggested a higher engagement of alternative pathway in respiration and a lower ATP production. The lower demand for ATP connected with inhibition of leaf growth may influence the ATP producing processes and ATP concentration. Thus, the ATP concentration in the leaves depends stronger on Pi content than on PN and RD.     

Adenosine Triphosphate-Dependent Calcium Uptake by Rat Submaxillary Gland Microsomes

Microsomes from rat submaxillary glands are able to take up calcium from the suspension media. Calcium uptake is greatly increased by the presence of ATP. This effect of ATP is not detected at 0°C. ADP cannot replace ATP to potentiate calcium uptake. ATP-dependent calcium uptake is not observed in the absence of magnesium. ATP-dependent calcium uptake is enhanced by oxalate and, to a lesser degree, by inorganic phosphate. Total calcium per milligram of microsomal protein observed when tests were performed without oxalate closely parallels the amounts for skeletal and cardiac muscles reported by several authors. Calcium uptake in salivary gland microsomes is slower than in muscle microsomes. Speculations are considered about the role of ATP-dependent calcium uptake. It is suggested that a decrease in intracellular free calcium levels returns these cells to the resting state after secretion.     

Effects of L-thyroxine on incorporation of (32)P into phospholipids of freshwater eels

The effects of L-thyroxine on phospholipid biosynthesis, via (32)P incorporation, were studied in gill, kidney, liver and muscle tissue of eels acclimatized at 11 degrees C. L-thyroxine treatment had no effect on tissue content of lipid, inorganic and organic acid-soluble phosphorus. Only an increase of the specific radioactivities of lipid, inorganic and organic acid-soluble phosphorus was observed in the muscle. Percentage distribution of (32)P among classes of phospholipid were significantly altered in liver and muscle, without change in phospholipid composition. A specific effect of L-thyroxine on (32)P incorporation into phosphatidic acid in muscle and liver has been shown. As expected by the higher specific radioactivity of muscle inorganic and organic acid-soluble phosphorus, the increased incorporation of (32)P into phosphatidic acid probably results from a higher specific radioactivity of muscle ATP phosphorus.     

Ca2+ uptake, Ca2+-ATPase activity, phosphoprotein formation and phosphate turnover in a microsomal fraction of smooth muscle

Vesicles capable of phosphate-stimulated calcium uptake were isolated from the microsomal fraction of the smooth muscle of the pig stomach according to a previously described procedure which consists in increasing the density of the vesicles by loading them with calcium phosphate and isolating them by centrifugation [Raeymaekers, L., Agostini, B., and Hasselbach, W. (1981) Histochemistry, 70, 139--150]. These vesicles, which contain calcium phosphate deposits, are able to accumulate an additional amount of calcium. This calcium uptake is accompanied by calcium-stimulated ATPase activity and by the formation of an acid-stable phosphoprotein. The acid-denatured phosphoprotein is dephosphorylated by hydroxylamine, which indicates that an acylphosphate is formed. This phosphoprotein probably represents a phosphorylated transport intermediate similar to that seen with the Ca2+-ATPase of sarcoplasmic reticulum of skeletal muscle. As with the Ca2+-ATPase of sarcoplasmic reticulum vesicles, this vesicular fraction catalyses an exchange between inorganic phosphate and the gamma-phosphate of ATP (ATP-Pi exchange) which is dependent on the presence of intravesicular calcium, and an exchange of phosphate between ATP and ADP (ATP-ADP exchange). The results further indicate that the turnover rate of the calcium pump, calculated from the ratio of calcium-stimulated ATPase activity to the steady-state level of phosphoprotein, is similar to that of Ca2+-ATPase of sarcoplasmic reticulum of skeletal muscle.     

Changes in inorganic phosphate and force production in human skeletal muscle after cast immobilization.

Cast immobilization is associated with decreases in muscle contractile area, specific force, and functional ability. The pathophysiological processes underlying the loss of specific force production as well as the role of metabolic alterations are not well understood. The aim of this study was to quantify changes in the resting energy-rich phosphate content and specific force production after immobilization. (31)P-magnetic resonance spectroscopy, three-dimensional magnetic resonance imaging, and isometric strength testing were performed in healthy subjects and patients with an ankle fracture after 7 wk of immobilization and during rehabilitation. Muscle biopsies were obtained in a subset of patients. After immobilization, there was a significant decrease in the specific plantar flexor torque and a significant increase in the inorganic phosphate (P(i)) concentration (P < 0.001) and the P(i)-to-phosphocreatine (PCr) ratio (P < 0.001). No significant change in the PCr content or basal pH was noted. During rehabilitation, both the P(i) content and the P(i)-to-PCr ratio decreased and specific torque increased, approaching control values after 10 wk of rehabilitation. Regression analysis showed an inverse relationship between the in vivo P(i) concentration and specific torque (r = 0.65, P < 0.01). In vitro force mechanics performed on skinned human muscle fibers demonstrated that varying the P(i) levels within the ranges observed across individuals in vivo (4-10 mM) changed force production by approximately 16%. In summary, our findings clearly depict a change in the resting energy-rich phosphate content of skeletal muscle with immobilization, which may negatively impact its force generation.